Bacterial Pathogenesis Research Group, Department of Microbiology, PO Box 53, Monash University, Victoria 3800, Australia1
Author for correspondence: Julian I. Rood. Tel: +61 3 9905 4825. Fax: +61 3 9905 4811. e-mail: Julian.Rood{at}med.monash.edu.au
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ABSTRACT |
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Keywords: Erm determinants, conjugative transposons, mobilization
Abbreviations: DR, direct repeat; MLS, macrolide-lincosamide-streptogramin B
The GenBank accession number for the Tn5398 element and flanking sequence is AF109075.
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INTRODUCTION |
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The C. difficile isolates that cause antibiotic-associated diarrhoea are usually not resistant to the antibiotic responsible for the onset of infection, suggesting that an important factor in pathogenesis is the elimination of the normal flora of the intestine by antibiotic therapy. The end result is the provision of an ecological niche for the germination and growth of C. difficile spores that originate in the hospital environment (Banerjee & LaMont, 2000 ). However, because of the strong association between antimicrobial therapy and the onset of C. difficile disease, antibiotic resistance in this organism has been extensively studied (Chow et al., 1985
; Levett, 1988
; Roberts et al., 1994
; Wüst & Hardegger, 1988
). In addition, studies have shown that resistance to clindamycin increases the risk of C. difficile-associated diarrhoea (Johnson et al., 1999
).
Clindamycin and erythromycin, both of which are members of the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics, have often been implicated in the onset of C. difficile-associated disease. The most common mechanism of resistance to these antibiotics involves N6-dimethylation of a specific adenine residue of the 23S rRNA molecule (Leclercq & Courvalin, 1991 ). This alteration of the antibiotic target site is invariably catalysed by an rRNA methyltransferase that is encoded by an erm gene. Numerous erm genes have been characterized and divided into distinct classes based on their level of sequence similarity (Roberts et al., 1999
). In general, each of the classes is loosely associated with a particular bacterial genus, with the exception of the Erm B class of determinants, which have been detected in a wide variety of bacterial genera, indicating their potential for intergeneric transfer.
Hybridization analysis has indicated that MLS-resistant strains of C. difficile carry erm(B) genes (Farrow et al., 2000 ; Hächler et al., 1987
). The Erm B determinant carried by C. difficile strain 630 has been shown to be transferred by a conjugation-like mechanism to C. difficile (Wüst & Hardegger, 1983
), Staphylococcus aureus (Hächler et al., 1987
) and Bacillus subtilis (Mullany et al., 1995
). Transfer has been shown to occur in the absence of detectable plasmid DNA. The B. subtilis transconjugants could transfer the Erm B determinant back to C. difficile, with integration of the determinant occurring at a specific site on the C. difficile chromosome. By contrast, integration was not site-specific in B. subtilis. Because of these observations it was proposed that the Erm B determinant from C. difficile resides on a conjugative transposon, Tn5398 (Mullany et al., 1995
). This element has not been completely sequenced or characterized, although we have shown (Farrow et al., 2000
) that it carries two identical erm(B) genes that are separated by a copy of the direct repeat (DR) sequence that is found on either side of the erm(B) gene from Clostridium perfringens (Berryman & Rood, 1995
). These C. perfringens DR sequences are two directly repeated segments of DNA that primarily consist of an ORF, ORF298, which shows low levels of identity at the amino acid level to Soj and ParA proteins, flanked by highly palindromic sequences, palA and palB. In C. difficile strain 630 the two erm(B) genes are separated by a copy of the DR sequence and are flanked downstream by a variant of this DR sequence from which ORF298 has been deleted and flanked upstream by a DR variant that contains only the sequence downstream of palB (Farrow et al., 2000
).
The aim of this research was to characterize the putative transposon and to examine the arrangement and distribution of both the erm(B) genes and Tn5398 in C. difficile strains from diverse geographical locations. The results showed that there was considerable genetic heterogeneity in the organization of the erm(B) gene region and that Tn5398 was an unusual genetic element in that it did not contain any discernible recombinase or mobilization genes.
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METHODS |
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DNA sequencing and computer analysis.
DNA sequencing was carried out using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit and analysed using an Applied Biosystems 373 DNA sequencer. Both DNA strands were sequenced using sequence-specific oligonucleotide primers. Nucleotide and amino acid comparisons were accomplished using the National Center for Biotechnology Information BLAST server at http://www.ncbi.nlm.nih.gov/BLAST/. The SOSUI program developed by the Mitaku Group, Department of Biotechnology, Tokyo University of Agriculture and Technology, Japan (http://sosui.proteome.bio.tuat.ac.jp/cgi-bin/sosui.cgi?/sosui_submit.html) was used to predict the structure of putative transmembrane proteins.
DNA dot blots.
DNA dot blots were performed by transferring 8 µg chromosomal DNA from each respective strain to a Hybond-N+ (Amersham Pharmacia Biotech) nylon membrane using a Minifold I Dot Blotter (Schleicher & Schuell). The DNA was cross-linked to the membrane by exposure to UV light at 312 nm for 3 min. The DNA was denatured by prehybridization for a minimum of 3 h in a solution containing SDS. The blots were then probed at high stringency with digoxigenin (DIG)-labelled probes prepared by PCR with the oligonucleotide primers listed in Table 2 as follows. The probes included a 688 bp erm(B)-specific probe (PCR primers: 2980 and 2981), a 399 bp ORF298-specific probe (4538 and 4451), a 339 bp palA-specific probe (4191 and 4537), a 984 bp ilvD-specific probe (6018 and 6278), a 933 bp hydD-specific probe (6339 and 6940), a 792 bp ORF13-specific probe (6019 and 6785), a 1166 bp effD-specific probe (9069 and 10237) and a 1124 bp ispD-specific probe (11546 and 11864). Following high stringency washes the blots were developed using the chemiluminescent substrate CDP-Star (Roche Molecular Biochemicals).
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Filter matings.
C. difficile cultures were grown overnight on BHIS agar at 37 °C. Single colonies of the donor and recipient were separately used to inoculate 20 ml BHIS broth and the cultures were grown until mid-exponential phase (OD600 0·45). The cells were then harvested by centrifugation at 1500 g for 10 min at room temperature and the cell pellets resuspended in 1 ml BHIS broth. Aliquots (100 µl) of the donor and recipient suspensions were mixed together on 0·45 µm pore-size nitrocellulose filters on BHIS agar. After incubation for 24 h at 37 °C, the filters were then removed, vigorously washed with 1 ml BHIS broth and 100 µl aliquots spread onto BHIS agar supplemented with the appropriate antibiotics and incubated for 48 h.
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RESULTS |
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In addition to duplicated erm(B)ORF3 genes and ORF298, which were previously identified by Farrow et al. (2000) , the gene region contained nine other potential genes (Fig. 1
). Upstream of the Erm B determinant two complete ORFs and one partial ORF were detected. The latter appeared to encode a protein with 52% amino acid sequence identity to IlvD from B. subtilis (Sorokin et al., 1996
) and had greater than 50% identity to IlvD proteins from many other organisms. IlvD is a dihydroxy-acid dehydratase that is involved in the synthesis of the amino acids isoleucine and valine.
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Downstream of the Erm B determinant there are three ORFs, designated ORF13, ORF9 and ORF7 (Fig. 1) because their putative products share 20, 43 and 20% amino acid sequence identity, respectively, to the equivalent ORFs from the conjugative transposon Tn916 from Enterococcus faecalis (Flannagan et al., 1994
). The function of these ORFs in Tn916 is not known.
The effR and effD genes were also located downstream of the Erm B determinant (Fig. 1). EffR shows low-level (1520%) identity to several repressor genes, including the repressor of the marRAB operon from Salmonella typhimurium, which is involved in multiple antibiotic resistance (Sulavik et al., 1997
). EffD showed some similarity to integral membrane proteins from a variety of organisms, including the hyperthermophile Pyrococcus horikoshii (Kawarabayasi et al., 1998
) and the alkaliphilic Bacillus sp. C-125 (Takami et al., 1999
). Computer analysis using the membrane prediction program SOSUI suggested that EffD was a membrane protein with 12 membrane-spanning helices.
The putative protein encoded by the gene located 3' of ORF7, ispD, had 4852% identity to intracellular serine proteases from B. subtilis (Koide et al., 1986 ), Bacillus amyloliquefaciens and Bacillus polymyxa (Surova et al., 1994
; Takekawa et al., 1991
). In B. subtilis the homologous protease ISP-1 has been postulated to have a critical role in sporulation, possibly through the turnover of intracellular proteins, the processing of spore coat protein precursors and the inactivation of transcarbamylase and several other enzymes (Koide et al., 1986
).
The last ORF identified in this study was designated flxD because it appeared to encode a protein with 3945% identity to flavodoxin proteins from Clostridium beijerinckii (Tanaka et al., 1974a ), Megasphaera elsdenii and Treponema pallidum (Fraser et al., 1998
; Tanaka et al., 1974b
). Flavodoxins are low-molecular-mass proteins that function as electron transfer agents in a variety of microbial metabolic processes (Simondsen & Tollin, 1980
).
Since ilvD, hydR, hydD, ispD and flxD were probably housekeeping genes we postulated that Tn5398 extended from a region downstream of hydD to the intergenic ORF7ispD region (Fig. 1). To examine this hypothesis a series of DIG-labelled probes specific for ilvD, hydD, erm(B), ORF13, effD and ispD were used in dot blots to examine chromosomal DNA from the wild-type C. difficile erythromycin-resistant strain 630, the susceptible C. difficile recipient CD37 and four independently derived transconjugants. The latter were derivatives of CD37 and were the result of conjugative transfer of erythromycin resistance from strain 630. If a particular gene was of a generic or housekeeping nature, we would expect a gene-specific probe to bind to DNA from all of the strains tested. If the gene was located on Tn5398 we would expect the probe to hybridize with DNA from the wild-type and the transconjugants but not the recipient. The results showed that each of the predicted housekeeping genes hybridized to all of the strains tested (Fig. 2
). By contrast, the erm(B), ORF13 and effD probes hybridized only to the wild-type and transconjugant strains, indicating that these genes are likely to be located within Tn5398.
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Variation in the genetic organization of erm(B) gene regions of C. difficile strains from different geographical locations
Prior to this study we had reported that the duplicated arrangement of the erm(B) genes in C. difficile strain 630 was novel when it was compared to Erm B MLS resistance determinants from other bacterial species (Farrow et al., 2000 ). To determine if this arrangement was common in C. difficile we analysed 27 erythromycin-resistant C. difficile strains from a range of geographical locations and clinical sources (Table 1
). DNA dot blots were carried out on chromosomal DNA from each isolate using an erm(B)-specific probe. The results showed that nine of these strains, including all five of the Japanese isolates, did not carry an erm(B) gene (Fig. 3
). Probing with an ORF298-specific probe showed that an additional four isolates contained an erm(B) gene but did not contain the complete DR sequence that contains ORF298 (Fig. 3
). Subsequent analysis, using a probe specific for the palA-like sequence that is located on either side of ORF298, revealed that the French isolate, 685, which did not contain ORF298, had a DR sequence with an internal deletion (Fig. 3
), as found previously in other bacteria (Berryman & Rood, 1995
; Farrow et al., 2000
).
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Three C. difficile isolates had the same arrangement as observed in strain 630 and appeared to have a complete copy of Tn5398. That is, they had two erm(B) genes, one located upstream of a complete DR sequence and the other upstream of an incomplete DR sequence, and had a genetically linked ORF13effD gene region (Fig. 5e). This group was geographically diverse as it included two Australian isolates from different hospitals and a French isolate, as well as the prototype Swiss isolate, strain 630.
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DISCUSSION |
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Tn5398 does not appear to encode genes that are involved in either excision, integration or conjugation. We have shown that it is 9·6 kb in size and encodes two copies of erm(B)ORF3 and one copy of ORF298 (Farrow et al., 2000 ). In addition, there is only one incomplete and four complete ORFs located within the putative transposon. The proteins encoded by the effR and effD genes are unlikely to be involved in the transposition of the putative transposon, as they appear to encode a potential efflux protein and its associated regulator. The remaining ORFs, ORF13, ORF9 and ORF7, have similarity to equivalent ORFs from the prototype conjugative transposon, Tn916. The ORF7 protein shows limited homology to sigma factors (Flannagan et al., 1994
). It has been postulated to have a regulatory role in the mobility of Tn916 because in the presence of tetracycline, increased ORF7 expression leads to increased transcription of ORF7, ORF8, xis, int and other genes (Celli & Trieu-Cuot, 1998
). The finding that the right end of Tn5398 is internal to ORF7 may have implications for the level of excision, transfer and integration of the element. After excision from the donor chromosome, Tn5398 would leave behind part of ORF7, resulting in an incomplete ORF7 gene in the circular intermediate. The result could be altered levels of transcription of other genes involved in transfer of the element. The end of ORF7 also appears to be the target sequence for the element in recipient strains such as CD37. It is this region of the ORF7 protein that has identity to the helixturnhelix motif of sigma factors, which is involved in DNA binding. Fusion with this region may provide a selective advantage for recombination of the circular intermediate at the target site.
In Tn916 ORF9 has been predicted to be a putative transcriptional repressor; however, the role of this repressor in the mobility of Tn916 has not been determined (Celli & Trieu-Cuot, 1998 ). The ORF13 protein has no known role in the mobility of Tn916. If these Tn916 homologues have any role at all in the movement of the Tn5398 element, it would appear that they are most likely to encode proteins that are involved in the regulation of transposition events, rather than proteins that are involved in excision, mobilization, transposition or integration.
ORF298 is the only other ORF that could encode a protein involved in Tn5398 mobility. The putative ORF298 protein has some similarity to replication proteins and proteins from the ParA and Soj families. ParA and Soj proteins generally have a role in the partitioning of plasmids and chromosomes during the replication cycle (Easter et al., 1998 ; Sharpe & Errington, 1996
). It appears unlikely that ORF298 has a role in either the excision or integration of Tn5398 but this possibility cannot be completely eliminated.
In addition to being capable of catalysing its own conjugative transposition, Tn916 is capable of enhancing the transfer of another homologous conjugative transposon that is co-resident in the cell (Flannagan & Clewell, 1991 ) and of mobilizing non-conjugative plasmids (Jaworski & Clewell, 1995
; Showsh & Andrews, 1999
). Based on its small size and our comparative analysis of the genes carried on Tn5398, we postulate that it is more likely to be a non-conjugative but mobilizable element rather than a conjugative transposon.
Mobilization of non-conjugative plasmids by Tn916 does not appear to be dependent on the presence of a functional mobilization, or mob, gene on the plasmid, but does require the presence of an origin of transfer, or oriT, sequence. It is postulated that the same protein or proteins involved in the nicking of the Tn916 circular intermediate at its oriT site nicks similar sequences present on co-resident mobilizable plasmids. Once nicked, the plasmid then assumes a relaxed form, which is capable of being transferred during conjugation (Showsh & Andrews, 1999 ).
In addition to Tn5398, strain 630 carries a second conjugative transposon, Tn5397 (Mullany et al., 1990 ), which is closely related to Tn916 (Roberts et al., 2001
). Comparison of Tn5397 and Tn916 reveals that they have very similar conjugation regions (ORF15ORF23), but different insertion and excision modules (Roberts et al., 2001
). In addition, the oriT sites present on both elements are identical and the putative ORF23 mobilization proteins have greater than 90% identity. Analysis of Tn5398 reveals the presence of two potential oriT sites, which are located within the coding sequence of ORF298 and in the intergenic space between ORF3 and ORF13 (Fig. 6
). The nic sites (TGGTGT) of these two potential oriT sites are identical to the nic sites found on Tn916 and Tn5397. We postulate that Tn5398 is excised from the chromosome either by TndX, the site-specific recombinase responsible for the excision of Tn5397, or by another large resolvase encoded on the chromosome of strain 630. The resultant circular intermediate of Tn5398 would then be nicked at one of the oriT sites by the same protein responsible for nicking the Tn5397 circular intermediate and subsequently transferred to a recipient cell by a Tn5397-dependent process. Once in the recipient the element may be either integrated into the chromosome by means of the TndX protein or by another large resolvase encoded on the chromosome of the recipient cell. Attempts to verify this hypothesis by using outward firing PCR primers to amplify the putative circular intermediate were unsuccessful. However, in the absence of a positive chromosomal control, little can be concluded from this experiment.
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Tn5398 is present in C. difficile strains from diverse sources
To determine if Tn5398, with its duplicated erm(B) genes, was common in C. difficile we carried out a series of comparative PCR and dot blot studies. The results showed that the arrangement of the Erm B determinants in 15 C. difficile isolates from diverse sources was not the same, with four major genetic variants being detected. The simplest variant was that of a single erm(B) gene and the most complex was represented by the Erm B determinant carried by Tn5398 (Fig. 5). Tn5398 was present in strain 630, which was originally from Switzerland, and three other C. difficile isolates, AM1180 and AM1185, isolated from different Australian hospitals, and the French isolate 660/2. The identification of these strains provides evidence that the duplicated erm(B) gene organization carried by Tn5398 is widespread and is therefore likely to be transferred between C. difficile isolates.
In general, each of the genetic variants was represented by isolates from a wide variety of geographical sources, with the exception of the group that carried a single erm(B) gene flanked by a downstream complete DR sequence (Fig. 5c). This group of isolates, with the exception of strains 662 and B1, were all isolated in the USA. With one exception, AM140, these USA isolates were isolated from large outbreaks of diarrhoea that occurred in four hospitals located in different parts of the country (Johnson et al., 1999
). Analysis of these strains by restriction digestion, PCR and PFGE had previously determined that these isolates were actually derivatives of the same strain (Johnson et al., 1999
), which was referred to as the epidemic strain. The isolates were all highly resistant to clindamycin and were shown to account for approximately 3066% of the C. difficile strains isolated at these hospitals during the period 1989 to 1992 (Johnson et al., 1999
). We previously showed that each of these isolates contained an erm(B) gene (Johnson et al., 1999
). Our finding that they contain Erm B determinants with the same genetic organization supports the conclusion that they are derivatives of an epidemic strain. It was previously suggested (Johnson et al., 1999
) that the erm(B) gene present in these isolates was associated with Tn5398. This study reveals that this is unlikely as the Tn5398-specific gene, effD, was not detected in these isolates.
We previously proposed that the C. perfringens Erm B determinant, which consists of an erm(B) gene flanked by two complete DR sequences, represents the Erm B progenitor and that Erm B determinants in other bacteria probably evolved by homologous recombination events that removed part of the DR sequences (Berryman & Rood, 1995 ). The different genetic arrangements of the various C. difficile Erm B determinants observed in this study are all consistent with this hypothesis. However, despite the fact that many erm(B) genes are located on mobile genetic elements that can move freely between different bacterial species, C. perfringens still appears to be the only species that contains an erm(B) gene associated with two intact copies of the DR sequence.
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ACKNOWLEDGEMENTS |
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Received 21 May 2001;
revised 2 July 2001;
accepted 3 July 2001.