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1 Division of Medical Microbiology, School of Infection and Host Defence, University of Liverpool, Daulby Street, Liverpool L69 3GA, UK
2 Division of Gastroenterology, School of Clinical Science, University of Liverpool, Crown Street, Liverpool L69 3BX, UK
Correspondence
Craig Winstanley
C.Winstanley{at}liv.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary tables showing the E. coli isolates used in this study and distribution of sequences according to PCR assays, the oligonucleotide primers used for PCR amplification, and the percentage distribution of PAIs amongst E. coli collections are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and inflammatory bowel diseases (IBDs), including Crohn's disease. Several independent groups have identified mucosa-associated Escherichia coli in Crohn's disease (Martin et al., 2004; Darfeuille-Michaud et al., 1998; Swidsinski et al., 2002; Ryan et al., 2004; Mylonaki et al., 2005; Kotlowski et al., 2007). Immunohistochemical studies have shown E. coli antigens within tissue macrophages in Crohn's disease tissue (Liu et al., 1995), and E. coli DNA can be isolated from the majority of Crohn's disease granulomas in tissue sections (Ryan et al., 2004). Similarly, in a prospective study of patients screened for colorectal cancer, intracellular bacteria, notably E. coli, were isolated from both tumour and distant histologically normal mucosa (Swidsinski et al., 1998).
In a previous study, we confirmed that Crohn's disease and colon cancer isolates of E. coli, particularly those lying beneath the colonic mucous layer, were more likely to adhere to and invade epithelial cell lines, and we showed that this property correlated well with their ability to agglutinate human red blood cells, regardless of blood group (Martin et al., 2004). In both Crohn's disease and colon cancer, we (Martin et al., 2004) and others (Swidsinski et al., 1998) have found mucosa-associated E. coli that resisted gentamicin treatment of the mucosal samples and were therefore presumed to be intracellular. It has been demonstrated that Crohn's disease-associated E. coli adhere to and invade epithelial cell lines in vitro (Darfeuille-Michaud et al., 2004; Martin et al., 2004) and replicate inside macrophage phagolysosomes (Bringer et al., 2006), with resulting giant cell formation (Meconi et al., 2007). It has been reported (Kotlowski et al., 2007) that these adherent and invasive E. coli (AIEC) belong to phylogenetic groups B2 and D (Clermont et al., 2000), typical of extraintestinal pathogenic E. coli (ExPEC), and that they exhibit a diffusely adherent pattern of adherence with Hep-2 cells, thus having many of the features of uropathogenic E. coli (UPEC). Like others, we have also shown that they lack the pathogenicity genes associated with E. coli that are typically pathogenic in the gut (Martin et al., 2004). Recently, it has been shown that AIEC of phylogenetic groups B2 and D can cause granulomatous colitis in boxer dogs (Simpson et al., 2006).
Thus far, genetic characterization of E. coli strains that occupy the submucosal niches in colon cancer and Crohn's disease has been very limited. Some progress has been made with the identification of genes responsible for adherence to and invasion of epithelial cells, but these studies have only been conducted in one ileal isolate from Crohn's disease, LF82 (Barnich et al., 2004; Rolhion et al., 2005, 2007).
In this study we describe the use of suppression subtractive hybridization (SSH) to characterize the genomic content of a mucosal E. coli colon cancer isolate and further analyse the distribution of subtracted sequences and UPEC-associated pathogenicity islands (PAIs) amongst a panel of mucosa-associated E. coli isolated from colonoscopic biopsies of patients with Crohn's disease and colon cancer, and of controls (patients with irritable bowel syndrome and sporadic polyps).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The bacteria were obtained after the overlying mucous layer had been removed by DTT treatment from biopsy samples taken from the sigmoid colon (Martin et al., 2004). The ileal isolate E. coli LF82 was a gift from Dr Darfeuille-Michaud, Laboratoire de Bacteriologie, Université d'Auvergne, Clermont-Ferrand, France. The K-12 derivative E. coli JM109 was used to obtain driver DNA for subtractive hybridization. The UPEC strains E. coli J96 and 536 (gift of Eva Moreno Pujol, Hospital Universitari Vall d'Hebron, Barcelona) were used as controls in PCR assays and, in the case of strain 536, for further subtraction work. All bacteria were maintained by growth on nutrient agar at 37 °C.
Construction and screening of subtraction libraries.
Genomic DNA was isolated from E. coli strains using the Wizard Genomic DNA Purification kit (Promega). SSH was carried out using the Clontech PCR-Select Bacterial Genome Subtraction kit as recommended by the supplier. In both hybridizations, DNA from E. coli HM229 was used as tester. DNA from E. coli strains JM109 and 536 was used as the driver in the first and second hybridizations, respectively. All DNAs were digested with RsaI. PCR amplicons obtained following SSH were cloned into pGEM-T (Invitrogen). The subtraction libraries of RsaI fragments thus constructed were screened by sequencing of plasmid DNA extracted from individual clones using M13 forward and reverse vector primers (Cogenics Lark). In order to identify genuinely subtracted sequences, BLASTN searches targeting the genomes of E. coli K-12 and 536 were conducted. Sequences sharing >90 % identity with the driver genome were omitted from further study. Sequences sharing <90 % identity with the genome of the relevant driver strain were further analysed using BLASTN and BLASTX searches of the general database. Similar BLASTN searches were used to determine the presence or absence of SSH sequences from the genomes of the UPEC strains CFT073 (Welch et al., 2002), 536 (Brzuszkiewicz et al., 2006) and UTI89 (Chen et al., 2006), and the enterohaemorrhagic E. coli (EHEC) strain EDL933 (Perna et al., 2001). All searches were done using the site http://www.ncbi.nlm.nih.gov.
PCR amplification screening of strains.
Oligonucleotide primers (Sigma-Genosys) for PCR amplifications are listed in Supplementary Table S2 along with the annealing temperatures used. DNA for PCR amplification was prepared by boiling a suspension of a few colonies in 5 % 200 µl Chelex-100 (Bio-Rad) for 5 min. After centrifugation, the top 150 µl was removed and stored at –20 °C. For PCR amplification, typically, 1 µl DNA was used directly in 25 µl volumes containing 1.25 U Taq DNA polymerase (Promega), 1x TaqMaster (Helena BioSciences), 300 nM each primer, 1x Taq buffer, 2.5 mM MgCl2 and 100 µM nucleotides (dATP, dCTP, dGTP, dTTP). Amplifications were carried out in an Eppendorf MasterCycler thermal cycler for 30 cycles consisting of 95 °C (1 min), annealing temperature (1 min) and 72 °C (2 min), with an additional extension time at 72 °C (10 min) following completion of the 30 cycles.
PCR assays for UPEC-specific PAIs have been described elsewhere (Sabaté et al., 2006; Johnson & Stell, 2000). In this study we used an amended version of the multiplex PCR assays described by Sabaté et al. (2006), whereby multiplex PCR B was split into two separate PCR assays consisting of assays for (1) PAI IIJ96 and PAI I536 and (2) PAI II536, PAI ICFT073 and PAI IJ96. Multiplex PCR A was used to assay for PAI III536, PAI IV536 and PAI IICFT073. Amplicon sizes and annealing temperatures are indicated in Supplementary Table S2. For the PAI IIJ96 and PAI I536 PCR assays, the extension time was increased to 3 min.
Oligonucleotide primers used in PCR assays for PAI VI536 were designed from SSH sequences obtained in this study. PCR assays for 229-4 and ECP1966 were multiplexed together. Phylogenetic groups were determined using assays published elsewhere (Clermont et al., 2000).
The SSH sequence 229-7 was extended by inverse PCR amplification. Genomic DNA from E. coli HM229 was digested with SalI, ligated using T4 DNA ligase and subjected to PCR amplification using the primers 229-7up (5'-TGCGCATGATTACCAGAC-3') and 229-7dn (5'-CGGTTCCTGATGTGTGCTA-3'). The resulting amplicon was sequenced using the same oligonucleotides as primers (Cogenics Lark). Further amplification and sequencing using the primers 5'-GGGCGATTTTTAGAAAGG-3' and 5'-TGCGCGTCATCAGCTTTC-3' was used to fill in the remaining gap and enable a complete ORF to be obtained.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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).
The sequences from a total of 116 SSH clones were obtained. Of the sequences, 85 (77 %) were genuinely subtracted from the genome of E. coli K-12, but 10 were repeated, leaving an output of 75 different SSH sequences. Of these, only one gave no significant match when used in a BLASTX search of the database. A summary of all SSH sequences obtained, organized according to their putative function by BLASTX match, is shown in Table 1.
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). Two of the SSH sequences (229-G3 and 229-C6) matched the known virulence-related proteins HlyD (involved in the transport of haemolysin A) and cytotoxic necrotizing factor 1 (CNF1), respectively. In the vast majority of cases, the BLASTX matches were with sequences that shared high levels of identity with sequences from the genomes of E. coli, Shigella, Yersinia or Salmonella. Notable exceptions to this were SSH sequence 229-H3, sharing only 40 % identity with an O antigen-related protein of Vibrio cholerae, and SSH sequence 229-7, which matched two adjacent genes from E. coli O157 : H7, but shared only 63 % BLASTX identity with one of these proteins, a putative adhesin/invasion-related protein (Table 1). Using inverse PCR amplification, we extended this sequence to obtain the whole of the putative gene (submitted to GenBank; accession no. EU046568). The predicted 441 aa protein shared 64 % identity with an IHP1-like protein from a Shiga toxin-producing E. coli (AAO83376).
Four of the SSH sequences matched hypothetical proteins encoded by an unpublished putative genomic island (AGI-1) of the avian pathogenic E. coli (APEC) strain BEN2908.
Using BLASTN searches, the SSH sequences were screened for their presence in three genome-sequenced UPEC strains (CFT073, 536 and UTI89) and a genome-sequenced EHEC strain (EDL933) (Table 1). Of the SSH sequences, 53 % were present in the genomes of one or more UPEC strains but absent from the EHEC strain. A further 27 % were present in the genomes of one or more UPEC strains and the EHEC strain. Sixteen per cent were absent from all four genomes. Only 4 % of SSH sequences were present in the genome of the EHEC strain but absent from the genomes of all UPEC strains. Amongst the SSH sequences were seven with BLASTX matches to putative proteins encoded by one of the 131 UPEC-specific genes identified by Lloyd et al. (2007).
The SSH sequences included sequences that matched parts of the UPEC strain 536 PAIs PAI I536, PAI III536, PAI IV536, PAI V536 and PAI VI536 (Dobrindt et al., 2002; Brzuszkiewicz et al., 2006; Hochhut et al., 2006) and the UPEC strain CFT073 PAIs PAI ICFT073 (Guyer et al., 1998) and PAI IICFT073 (Rasko et al., 2001).
Identification of sequences present in the genome of E. coli HM229 but absent from the genome of UPEC strain 536
Our findings from the initial subtraction suggested that the accessory genome of E. coli HM229 shares more in common with UPEC than with intestinal pathogenic E. coli. In an attempt to enrich for genes within the accessory genome that might be specific to bacteria occupying mucosal regions of colon cancer or Crohn's disease, we carried out a second subtraction using the genome-sequenced UPEC strain 536 as the driver.
A total of 100 sequences were obtained, of which 62 were genuinely subtracted from the genome of strain E. coli 536. Four sequences were repeated, leaving a total of 58 different subtracted sequences. Of these 58 SSH sequences, 27 matched neither the UPEC strains UTI89 or CFT073 nor the EHEC strain EDL933 (Table 2). Of these 27, eight had a best BLASTX match against E. coli APEC O1, two had a best BLAST match against E. coli K-12, and five mainly O antigen/capsule-related SSH sequences had a best BLASTX match against other E. coli strains. A further eight SSH sequences matched outside the genus Escherichia. These included a putative SecA-related protein from V. cholerae.
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. Notably, there was a group of isolates from colon cancer (30–40 %) that tested PCR-positive for the UPEC PAIs PAI IIJ96, PAI I536, PAI II536 and PAI IICFT073. In contrast, one of six control isolates and no Crohn's disease isolates were PCR-positive for these PAIs, with the exception of one Crohn's disease and two control isolates testing PCR-positive for PAI IICFT073. The PCR assays for four different sequences from PAI VI536 indicated considerable variability in the carriage or nucleotide sequence of this island. Whilst some isolates were PCR-positive for all four of the sequences, others were PCR-positive for none, one, two or three of the sequences (Supplementary Table S1). The proportion of isolates that were PCR-positive for all four sequences was greater amongst the colon cancer isolates than either the Crohn's disease isolates or the controls (Table 3).
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).
It has been reported previously that the P-fimbriae-associated papC is present in many of the mucosal isolates (Martin et al., 2004). Since the PCR assays based on the study of Sabaté et al. (2006) were only designed to identify one papG allele type, we used further PCR assays to identify other papG allele types. The most common was papG allele II (Table 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Schmidt & Hensel, 2004; Gal-Mor & Finlay, 2006). E. coli is one of the best-studied examples of a bacterial pathogen that exhibits diversity in virulence due to the carriage of specific combinations of virulence genes and PAIs (Kaper et al., 2004). In particular, several groups have sought to identify and characterize UPEC-specific PAIs (Lloyd et al., 2007; Welch et al., 2002; Chen et al., 2006; Oelschlaeger et al., 2002a; Brzuszkiewicz et al., 2006). In the study of Brzuszkiewicz et al. (2006), the 536 islands are described as either 536-specific (for PAI I536 and PAI V536) or as part of the ‘common ExPEC gene pool’ (for PAI II536, PAI III536, PAI IV536 and PAI VI536; although some genes from PAI II536 and PAI III536 fall into the ‘536-specific’ category).
A simple phylogenetic scheme based on the presence or absence of a few selected genes has been used to demonstrate genetic differences between intestinal E. coli and ExPEC (Clermont et al., 2000). Several studies have noted the predominance of phylogenetic groups B2 and D amongst UPEC isolates (Johnson et al., 2005; Houdouin et al., 2006; Sabaté et al., 2006; Bidet et al., 2007) or meningitis isolates (Bidet et al., 2007; Ewers et al., 2007). However, few studies have sought to characterize E. coli isolates associated with IBD or colon cancer. Kotlowski et al. (2007) identified groups B2 and D as being of high prevalence in Crohn's disease, and they also screened Crohn's and ulcerative colitis isolates for the presence of multiple virulence genes, including adhesins. Interestingly, none carried cnf1/hlyA, an observation supported by our study with respect to Crohn's disease isolates.
Nevertheless, we found a group of colon cancer isolates that carried not only the cnf1/hlyA associated with PAI IIJ96 but also several other UPEC-associated islands. Interestingly, the I9/I10 putative fimbrial chaperone of PAI I536 and the ORF1 (ECP4571) region of PAI II536 match only the genomes of UPEC strains 536 and UTI89 when used to search the database. In addition, the CFT073 ferric enterobactin-transport protein/siderophore of PAI IICFT073 matches sequences from only four UPEC strains in the database. Thus, our distribution study indicates the presence amongst the colon cancer isolates of a group of E. coli isolates that carry multiple genes previously categorized as UPEC-specific. However, it has been reported that the accessory genome of a commensal E. coli strain which is a clinically safe, efficient colonizer of the gut also contains cnf1, hlyA and PAI II536 (Hejnova et al., 2005). Those authors suggested that these ‘virulence-associated’ genes might actually be contributing to fitness or colonization. It is worth noting that although related in the content of their accessory genome, isolates HM213, HM229 and HM334 are different according to typing by PFGE (Martin et al., 2004) and flagellin gene sequence (data not shown).
A closer analysis of the genome of a representative of this group, strain HM229, using SSH against E. coli K-12, indicated that the accessory genome of this strain was much more closely related to the genomes of UPECs than to those of intestinal E. coli. However, some subtracted sequences were not UPEC-associated. Four of the SSH sequences matched the E. coli O157 : H7 bacteriophage PhiV10, which has been completely sequenced (GenBank accession no. DQ126339), but for which there is no publication. Three further SSH sequences matched integrases, including one (SSH229-26) that matched a P4-like PAI-associated integrase similar to those implicated in playing an important role in the plasticity of E. coli genomes (Hochhut et al., 2006; Manson & Gilmore, 2006).
Further subtraction against the UPEC strain 536 enriched for sequences matching mobile genetic elements, other ExPEC strains such as APEC O1, and other UPEC strains or commensals, rather than strains associated with gastrointestinal disease. It has been reported elsewhere that the genome sequence of the avian pathogenic E. coli strain O1 : K1 : H7 shares strong similarities with human ExPEC genomes (Johnson et al., 2007). Two SSH sequences matched KpsD, a group 2 capsular polysaccharide production protein from a bacteraemia isolate (CP9). Group 2 capsule operons consist of highly conserved regions (e.g. kpsDMTE) that encode transport and assembly functions, combined with highly variable type-specific regions that encode the synthesis and/or polymerization of the specific component sugars of the particular polysaccharide (Whitfield & Roberts, 1999).
Sabaté et al. (2006) compared 100 UPEC and 50 commensal E. coli isolates, according to single PCR assays for each of eight UPEC-associated PAIs. Overall, 93 % of UPEC isolates carried PAIs, compared to 40 % of commensal isolates. However, it is interesting to compare the percentage carriage of individual islands between the isolate sets used in our study and that of Sabaté et al. (2006). The percentage carriage of many PAIs was very similar between isolates from UPEC (Sabaté et al., 2006) and colon cancer (Supplementary Table S3). It is also interesting to note that isolates carrying the same number of PAIs generally carry the same combinations of PAIs. This tendency towards specific combinations of PAIs has been reported before for UPEC and commensal isolates (Sabaté et al., 2006), and is indicative of a stepwise sequential programme of evolution rather than a random acquisition of PAIs.
Based on four PCR assays alone, we found some evidence for variations in PAI VI536 amongst the isolates in our panel. Instability in UPEC PAIs has been demonstrated before (Middendorf et al., 2004). Again, isolates sharing the same number of PCR-positives for the four assays had the same combination of PCR-positives. Thus, even within a single PAI, there is evidence for sequential evolution.
PAI IV536 is the most widely distributed of the islands amongst commensal and UPEC isolates (Sabaté et al., 2006). This island is related to the so-called high-pathogenicity island (HPI), which is widespread amongst the Enterobacteriaceae (Schubert et al., 2004).
It has been demonstrated that the ability of UPEC to cause symptomatic infection is enhanced by adhesins, including type 1 and P fimbriae (Klemm & Schembri, 2000; Oelschlaeger et al., 2002b). The SSH analysis of strain HM229 identified sequences from both type 1 and P fimbriae. P fimbriae, encoded by the pap operon, enhance the establishment of bacteriuria, activate the innate immune response (Wullt et al., 2000, 2002; Bergsten et al., 2004), and are widely distributed in UPEC and APEC (Johnson & Stell, 2000; Rodriguez-Siek et al., 2005). Binding is mediated by the tip adhesin PapG, which has at least three allelic forms, of which allele II is the most common (Johnson et al., 1998; Johnson & Stell, 2000). papC was widely distributed amongst the mucosal isolates from colon cancer (7/10) and Crohn's disease (4/8), with the relative distributions of the papG alleles resembling those reported elsewhere for UPEC (Johnson & Stell, 2000; Houdouin et al., 2006). However, for three papC PCR-positive isolates we did not get a PCR-positive for any of the papG alleles [including the rare papGIa allele variant (Johnson & Stell, 2000) (data not shown)], suggesting further allelic variation. An association between papG allele III and cnf1 has been reported elsewhere for UPEC (Johnson & Stell, 2000). However, of the three cnf1-positive isolates in this study, only one carried papG allele III.
The SSH analysis also identified other putative gene/protein sequences with predicted roles in adherence, invasion or haemagglutination. The SSH sequence 229-29 shared similarity with large proteins implicated in haem utilization or adhesion in UPEC. The SSH sequence 229-7 carries a YadA-like C-terminal region. YadA is the major adhesin of Yersinia enterocolitica, and is essential for establishing infection by mediating adhesion to host cells and conferring resistance against bactericidal activity. We made a knockout mutant for the full ORF associated with SSH sequence 229-7. However, we could identify no change in either adherence or invasion with I407 or Caco-2 cell lines, nor could we detect any difference in haemagglutination (data not shown). This may be because these are multifactorial phenotypes. For example, UPEC carry multiple adhesion-related genes, but carriage of individual adhesion-related genes, such as papC, can be well below 100 % (Johnson & Stell, 2000; Rodriguez-Siek et al., 2005; Houdouin et al., 2006).
PCR screening of Crohn's disease and cancer E. coli isolates for the presence of sequences 229-7 and 229-29 suggested a distribution very similar to that of cnf1 (Supplementary Table S1). In particular, the colon cancer isolates HM213, HM229 and HM334 carry cnf1, 229-7 and 229-29. The exotoxin CNF1 is an acknowledged virulence factor of UPEC but its prevalence has been reported as 16–27.5 % of UPEC isolates (Johnson & Stell, 2000; Rodriguez-Siek et al., 2005). Hence, the carriage rates for our colon cancer isolates for this UPEC-associated virulence factor are very similar to the reported figures for UPEC isolates. In contrast, either none or only one of the Crohn's disease isolates carried many of the pathogenicity-related genes.
The possible role of mucosally adherent E. coli in colon cancer pathogenesis is currently speculative, but there is growing interest in the possible role of inflammation (Rhodes & Campbell, 2002), and perhaps particularly in NF
B activation (Greten et al., 2004), in colon cancer. Bacterial adhesion, perhaps to dysplastic mucosa lacking an overlying mucus coat, has the potential to induce epithelial cell changes that could promote cancer development (Hope et al., 2005).
It is clear from our study and others that neither the colon cancer nor the Crohn's disease mucosal E. coli populations are uniform. However, it is possible that a subset of isolates plays an important role in the pathogenicity of these diseases. We identified a subset of colon cancer isolates, not represented amongst the Crohn's disease isolates, that carry multiple UPEC-associated virulence genes. The clinical relevance of this group remains unknown, but the presence of such strains merits further investigation.
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ACKNOWLEDGEMENTS |
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We would like to dedicate this paper to the memory of Professor Tony Hart, an inspirational colleague who is sadly missed by us and by many others in the worldwide microbiological community.
Edited by: D. L. Gally
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Received 10 September 2007;
revised 17 October 2007;
accepted 2 November 2007.
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