INTRODUCTION

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Bacteroides fragilis is the anaerobic microorganism most frequently isolated from infectious processes in humans (6) as well as a common component of the normal flora of the colon (7). Some of the strains belonging to this species are able to produce an enterotoxin and are therefore termed enterotoxigenic B. fragilis (ETBF) strains (17). ETBF strains are responsible for diarrheal diseases in young farm animals, including lambs, calves, and foals (17-19). Several studies have also shown an association between the isolation of ETBF from feces and acute diarrhea in children 1 to 5 years old (27, 28, 30). In addition, ETBF can be recovered from the normal fecal flora of healthy subjects, especially adults (25). In some of the subjects harboring ETBF, the enterotoxin is present in the feces in a biologically active form and can be detected by a cytotoxicity assay (26).

B. fragilis enterotoxin, recently termed fragilysin (20), has been characterized as a 20-kDa zinc-dependent metalloprotease belonging to the metzicins family, precisely to the subfamily that comprises the eukaryotic collagenases or matrixins (15). Recently, the target of fragilysin has been identified as the cell surface protein E-cadherin, which is the principal structural component of the zonula adherens and is responsible for cell-cell adhesion of eukaryotic cells (32).

Two groups of investigators have independently cloned and sequenced the enterotoxin gene from two ETBF strains and have identified two different allelic forms of this gene. The first published sequence of the enterotoxin gene, bftP (or bft-1), was obtained from strain VPI 13784, a lamb isolate (12). The other isoform, bft-2, was sequenced from a porcine isolate (8). Both isoforms code for a protein much larger than the mature enterotoxin, a "preprotoxin" of 45 kDa comprising a signal peptide and a protoxin from which the mature toxin is released upon cleavage. Although the two forms of the enterotoxin gene are 95% identical, they show lower homology in the region corresponding to the mature toxin moiety (8). New allelic forms of the enterotoxin gene were found in ETBF isolated from blood in Korea (5) and in fecal isolates from Japan (N. Kato, Final Program of the 2nd World Congress on Anaerobic Bacteria and Infections, abstr. 5.002, p. 74, 1998) and were termed Korea-bft and bft-3, respectively. As the corresponding nucleotide sequences are identical, we refer to the third allelic form of the enterotoxin gene as bft-3 throughout this paper. The bft-3 isoform shares high similarity with the other two forms but is more related to bft-2 (96% identity) (5). Its frequency among ETBF strains outside East Asia remains unknown.

Moncrief et al. (16) found that the B. fragilis enterotoxin gene is located in a pathogenicity islet together with a putative second metalloprotease (MP II) gene which has a low identity (28%) with the enterotoxin gene. The pathogenicity islet of B. fragilis is a genetic unit that shares some characteristic features with the larger pathogenicity islands present in pathogenic strains of Escherichia coli, Salmonella, or Helicobacter pylori. It contains virulence genes (for the enterotoxin and the putative virulence factor MP II), has a lower G+C content than the B. fragilis chromosome, contains almost identical direct repeats in proximity to its ends, and is inserted in the same chromosomal region in different strains (16).

In a previous study, we used PCR to amplify a portion of the bft gene and showed that this gene is present in all ETBF strains but not in nonenterotoxigenic strains (24). In the present study, using PCR-restriction fragment length polymorphism (RFLP) analysis, an allele-specific PCR, and sequencing data, we have shown the frequency of the two principal isoforms, bft-1 and bft-2, in a large collection of ETBF strains of human origin. We have also found that the variant bft-3, although rare, is present in ETBF isolated in Europe. Moreover, we have found a duplication of both the bft and MP II genes in five strains that probably represents a duplication of the entire islet.

	MATERIALS AND METHODS

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Bacterial strains. Sixty-eight ETBF strains from different sources were studied; these comprised 28 strains isolated from clinical (extraintestinal) samples, 23 strains isolated from the feces of children (18 with diarrhea), and 17 strains isolated from the feces of adults (10 with diarrhea). The extraintestinal isolates came from the collection of the Istituto Superiore di Sanità and included isolates obtained from Italy (18 strains), the United Kingdom (6 strains), and the United States (4 strains) and characterized in previous studies (22, 23). The fecal isolates were obtained in Italy during previous (22, 25) and ongoing studies on the prevalence of ETBF in the country and were isolated in different geographical areas over a span of 5 years. The reference strain ATCC 43858, originally isolated in the United States from the feces of an infant with diarrhea, was also included. The bacterial strains were identified as ETBF by previously described methods, including a cytotoxicity assay with HT-29 cells (22) and PCR amplification of an internal fragment of the enterotoxin gene (24). Strain VPI 13784 (a gift from T. D. Wilkins, Virginia Polytechnic Institute and State University, Blacksburg) was included in the study as the prototype of the bft-1 genotype. The nontoxigenic strain B. fragilis NCTC 9343 was used as a negative control.

Chemicals and enzymes. Nylon transfer membranes (Hybond N) were obtained from Amersham International (Little Chalfont, Buckinghamshire, United Kingdom). Restriction endonucleases were purchased from New England Biolabs (Beverly, Mass.) and Roche Molecular Biochemicals (Milan, Italy). For PCR amplification, DynaZyme II (obtained from Finnzyme, Oy, Finland) or AmpliTaq (obtained from PE Applied Biosystems, Roche Molecular Systems, Branchburg, N.J.) DNA polymerase was used. The oligonucleotide primers were synthesized at Laboratori Genenco, M-Medical, Florence, Italy. Electrophoresis-grade agarose and low-melting-point agarose were purchased from Gibco BRL (Life Technologies Italia, San Giuliano Milanese, Milan, Italy); NuSieve was obtained from FMC BioProducts (Rockland, Maine); and agarose D-5, used for pulsed-field gel electrophoresis (PFGE), was obtained from Hispanagar (Burgos, Spain). Proteinase K and lysozyme were obtained from Roche. All other reagents and chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.). The media for bacterial growth were obtained from Oxoid (Basingstoke, United Kingdom).

PCR-RFLP analysis. ETBF strains were processed for PCR amplification as previously described (24). Briefly, the bacterial cells were boiled for 10 min, centrifuged at 13,000 × g for 5 min, and stored at 20°C until used as templates (2 µl in each PCR). All PCRs were performed with a GeneAmp PCR System 9600 (Perkin-Elmer) and a reaction volume of 100 µl, containing a 0.5 µM final concentration of each oligonucleotide, 200 µM each deoxynucleoside triphosphate, and 2.5 U of DynaZyme II Taq polymerase. Each amplification was preceded by 5 min at 94°C and consisted of 35 cycles of 60 s at 94°C, 60 s at 56°C, and 120 s at 72°C, followed by a final 5 min at 72°C.

Allele-specific PCR. In order to distinguish between bft-2 and bft-3, we devised allele-specific PCR assays. For bft-2, the primers used were BF5 and BFT2R (Table 1). BFT2R was designed based on the oligonucleotide sequence specific for bft-2, mapping in a gene region divergent from both bft-1 and bft-3, according to Franco et al. (8). For bft-3, the pair used was BF5-BFT3R (Table 1); BFT3R was designed in the same position as BFT2R but with a 2-base substitution at the 3' end, according to the sequence of bft-3 (5). The PCR conditions used were the same as those described for PCR-RFLP analysis.

Sequencing of the bft gene. The portion of the enterotoxin gene corresponding to the whole mature moiety was amplified using the primer pair BFTF-BFTR. These primers were designed based on consensus regions for the three isoforms (Table 1).

DNA extraction and Southern hybridization. B. fragilis cells were pelleted from 5 ml of an overnight culture in Wilkins-Chalgren broth. Chromosomal DNA was extracted as follows. Lysis was performed with 50 mM Tris-50 mM EDTA buffer (pH 8.0) containing 50 mM glucose, 5 mg of lysozyme per ml, 40 µg of proteinase K per ml, and 10 µg of RNase A per ml at 37°C for 30 min. The DNA was extracted three times with phenol-chloroform, the aqueous phase was collected, and the DNA was recovered by ethanol precipitation and resuspended in water. The DNA was digested with restriction enzymes according to the manufacturers' recommendations, run on an 0.8% agarose gel at 30 V for 20 h, and transferred to a nylon membrane by capillary blotting.

PFGE. For the preparation of genomic DNA suitable for PFGE, ETBF strains were grown in 10 ml of Wilkins-Chalgren broth to late log phase (optical density at 600 nm, 0.8 to 0.9). The bacterial cells were placed in agarose plugs and lysed by standard procedures (13).

	RESULTS

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Detection of the enterotoxin gene alleles. With the primer pair BF5-BF6, the expected product of 976 bp was amplified from all of the ETBF strains examined. The nonenterotoxigenic strain NCTC 9343 yielded no amplification product (data not shown).

View larger version (42K): [in this window] [in a new window]   FIG. 1.   Agarose gel electrophoresis of the bft gene PCR products of ETBF strains and of the RFLPs obtained with DraI or AccI digestion. Lanes: 1, strain VPI 13784; 2, ATCC 43858; 3, PR 838; 4, MT 2. U, unrestricted PCR products; D, PCR products digested with DraI; A, PCR products digested with AccI. M, molecular size markers. In accordance with the fragment lengths indicated in the text, the isoforms bft-1 and bft-2 or bft-3 can be distinguished in lanes 1 and 3 and in lanes 2 and 4, respectively. The lower-molecular-size fragments are not visible. By sequence analysis and isoform-specific PCR, ATCC 43858 was shown to harbor bft-2 and MT 2 was shown to harbor bft-3.

View larger version (104K): [in this window] [in a new window]   FIG. 2.   Agarose gel electrophoresis of the amplification products obtained with allele-specific PCR for the isoforms bft-2 and bft-3. Lanes: 1, VPI 13784; 2, ATCC 43858; 3, B 115; 4, MT 2; 5, UK 5312. M, molecular size markers.

Distribution of the enterotoxin gene isoforms. Among the ETBF isolates examined, bft-1 was found to be more common than bft-2, as it was detected in two-thirds of the strains. The bft-1 isoform was found in almost all isolates from the feces of adults, including individuals with and without diarrhea; only 1 strain out of a total of 17 was found to harbor the bft-2 isoform. However, the bft-2 isoform was as common as the bft-1 isoform in strains isolated from the feces of children. The association between isoform bft-2 and strains from children is statistically significant compared to that between bft-2 and adult strains (P, 0.0064) (Table 2). When strains isolated from children with diarrhea (nine strains bearing bft-1 and nine bearing bft-2) are compared to strains isolated from adults with diarrhea (nine strains bearing bft-1 and one bearing bft-2), the association is statistically significant (P determined by the Fisher exact test, 0.04). We found that the bft-3 isoform, originally described for isolates from Korea and Japan, also was present in our collection of strains originating from Europe and the United States, although much less frequently than the other two isoforms. Strain MT 2 was isolated from the stools of a healthy 4-month-old Italian baby in 1994; strain UK 5312 was originally isolated from a blood culture in the United Kingdom.

Southern analysis of the bft and MP II genes. We analyzed 16 ETBF strains by Southern hybridization: 10 harboring bft-1, 4 harboring bft-2, and 2 harboring bft-3 (Table 3). The intent was to hybridize the chromosomal DNA with probes for the bft and MP II genes to define whether these two genes were consistently associated in all strains, irrespective of the isoform borne. Prior to the experiments, controls were run to verify that the two probes were specific for the respective genes and that there was no cross-hybridization.

View larger version (69K): [in this window] [in a new window]   FIG. 3.   Southern hybridization analysis of B. fragilis chromosomal DNA digested with EcoRI. DNA was hybridized with the bft probe (A) or with the MP II probe (B). Lanes: 1, VPI 13784; 2, ATCC 43858; 3, MT 2; 4, AN 209; 5, PR 838; 6, B 45; 7, PR 823; 8, LZ 4; 9, NCTC 9343 (negative control, nontoxigenic strain). The positions of the molecular size standards are indicated.

View larger version (84K): [in this window] [in a new window]   FIG. 4.   PFGE restriction patterns generated by NotI digestion (A) and Southern hybridization with the bft probe (B) of ETBF chromosomal DNA. Lanes: 1, VPI 13784; 2, ATCC 43858; 3, MT 2; 4, AN 209; 5, PR 838; 6, B115; 7, PR 823; 8, B 69; 9, LZ 4. Lanes M1 and M2, molecular size markers. The hybridization pattern obtained with the MP II probe was identical to that obtained with the bft probe.

Cytotoxin production in an HT-29 cell assay. In an attempt to find an association between the cytotoxicity titer and the bft allele or the copy number of the bft gene, repeated determinations of cytotoxicity titers in HT-29 cells were obtained for 16 ETBF strains. In strains bearing either bft-1 or bft-2, the titers varied from 2 to 3 log₁₀ units. Both strains bearing bft-3 showed low toxin titers (1.6 and 2 log₁₀ units). Four out of five strains harboring a duplication of the bft gene (three strains bearing bft-1 and one strain bearing bft-2) were found to have cytotoxicity titers in the high range of the series. The only exception was the strain bearing double copies of bft-3, which had a low toxin titer (Table 3). Due to the limited number of strains examined, it was not possible to demonstrate statistically significant differences.

	DISCUSSION

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Although ETBF strains have been associated with diarrhea in children under 5 years of age (27, 28, 30), the pathophysiological mechanisms linking enterotoxin production to diarrhea are not completely understood. B. fragilis toxin, or fragilysin, cannot be considered a classical enterotoxin, being a metalloprotease similar to eukaryotic collagenases (15). In vitro and in vivo studies have shown that this toxin is able to damage the intestinal mucosa (both ileal and colonic) of various animal species, including humans (21, 26, 29), and to elicit fluid accumulation through increased permeability (26) and active chloride secretion (4). Recently, the zonula adherens protein E-cadherin has been recognized as the substrate for B. fragilis enterotoxin. It has been hypothesized that cleavage of the extracellular domain of E-cadherin can lead to alteration of the cytoskeletal structures and to increased intestinal permeability. This hypothesis represents a novel mechanism of action for a bacterial enterotoxin (32).

The presence of ETBF in the gut does not necessarily indicate disease, as the organism has been frequently isolated from the feces of healthy individuals. Asymptomatic carriage is particularly high in adults, but children also can harbor both the microorganism and the toxin without intestinal disturbances (25). This finding suggests that other factors that can be related to either the host or the microorganism are necessary for the development of diarrhea. A recent study has shown unresponsiveness of the colon mucosa of some subjects to B. fragilis toxin (29).

ETBF can produce different isoforms of the enterotoxin, the most common being those encoded by the bft-1 and bft-2 genes (8, 12). Although these isoforms display the same biological activities, Wu and coworkers have suggested that their potencies might be different (32). We examined several ETBF strains isolated from different sources (extraintestinal infections and feces of adults and children) by PCR-RFLP analysis to distinguish between the isoforms bft-1 and bft-2 or bft-3 and subsequently by an isoform-specific PCR to distinguish between bft-2 and bft-3. We found that the majority (65%) of the strains investigated harbor the bft-1 isoform. This allele is largely predominant in strains from the feces of adults (with or without intestinal symptoms), while strains isolated from children harbor either bft-1 or bft-2. The rarity of the isoform bft-2 in adults indicates that strains bearing this isoform are more apt to colonize (and consequently induce diarrhea in) children than adults. The factors responsible for this association are unknown, but they might consist either of different properties of the bft-2 toxin itself or of characteristics of the strains which allow better proliferation in the colon of children than in that of adults.

By sequence analysis and an allele-specific PCR, we found that two strains in our collection harbor the isoform bft-3, originally described for blood isolates from Korea (5) and for fecal isolates from Japan (Kato, Final Program of the 2nd World Congress on Anaerobic Bacteria and Infections). The two strains in our collection were isolated in the United Kingdom and in Italy; this finding indicates that the bft-3 allele, although rare, is present in geographical areas outside East Asia.

Interestingly, the sequences of the bft genes examined were 100% identical to the published nucleotide sequences of the three alleles, without a single base substitution. Although this observation is limited to the portion of the genes coding for the mature toxin, this lack of variation suggests that bft is a recent acquisition of B. fragilis, as already proposed by Smith and Callihan (31), and that each isoform is conserved because of an evolutionary advantage. The amino acid substitutions among the three deduced proteins are not abundant: bft-2 toxin diverges from bft-1 toxin in 25 amino acids out of 186, and bft-3 (which is more similar to bft-2 than to bft-1) diverges from bft-1 in 20 amino acids and from bft-2 in 8 amino acids. However, these substitutions cluster in two regions adjacent to the active site of the metalloprotease and the zinc-binding motif (8). Therefore, the three variants could exhibit subtly different receptor-binding preferences that could result in differences in host range and/or pathogenic potential. Similar differences have been described for other allelic proteins. For instance, the alleles speA1 and speA3 of Streptococcus pyogenes code for toxins which differ only in one amino acid; however, the product of speA3 has significantly greater mitogenic activity and affinity for the class II major histocompatibility complex and is associated with clinical cases of streptococcal toxic shock syndrome (11). The papG alleles of Escherichia coli, which code for variant forms of the P adhesin, are associated with different clinical syndromes, such as pyelonephritis and cystitis (10).

In our collection, the two strains carrying bft-3 were found to produce low levels of biologically active toxin in the HT-29 cell cytotoxicity assay. One explanation is that a smaller amount of the protein is produced. An alternative possibility is that the bft-3 toxin is less active on the cell system used, although Chung et al. have demonstrated that the purified bft-3 toxin from Korean isolates cleaves E-cadherin at a concentration similar to that observed with the other purified enterotoxins (5).

We cannot rule out the possibility that other enterotoxin variants with substitutions in areas not explored by the RFLP and PCR assays used in this study exist. Sequencing of more strains from different sources and geographical regions is necessary for a comprehensive analysis of the frequency of the different enterotoxin alleles.

Following the recent discovery of the pathogenicity islet of B. fragilis, which includes the fragilysin gene, B. fragilis has been added to the growing list of microorganisms carrying pathogenicity islands. This islet is much smaller than classical pathogenicity islands. It comprises only the enterotoxin gene and the MP II gene and lacks the genes coding for secretion systems necessary for the delivery of the toxin directly to target cells (14). However, the B. fragilis islet shares with the larger structures the property of transforming a typical commensal organism into a virulent organism (9).

An unexpected finding was that some ETBF strains possess double copies of the enterotoxin gene, associated with double copies of the MP II gene and possibly of the entire islet. Bacteria harboring more than one pathogenicity island have been described before: for instance, in the same strain of uropathogenic E. coli serotype O4, PAI I and PAI II are present together. However, the virulence genes carried by the two islands are different (2). In some Helicobacter pylori type I strains, the cag pathogenicity island appears as two regions separated by a long stretch of chromosomal DNA, containing different genes and probably derived from rearrangement driven by insertion sequences (3).

For B. fragilis, the whole islet, including the enterotoxin and MP II genes, appears to have undergone duplication in some strains, as deduced from Southern analysis; consequently, the two islets carry the same enterotoxin gene isoform, as confirmed by PCR-RFLP and sequencing. The islet duplication appears to be quite stable, as the Southern hybridization patterns were reproducible when the strains were examined after storage and repeated subculturing. Interestingly, with the exception of the bft-3 strain, the cytotoxicity titers obtained from supernatants of strains with double copies of the gene were among the highest in our series; however, the limited number of strains examined does not allow conclusions to be drawn.

Our observations indicate that there are complex microbiological properties of ETBF that need to be elucidated for a better understanding of the pathogenic potential of this microorganism.