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Previous Article | Next Article 
Infection and Immunity, February 2004, p. 684-690, Vol. 72, No. 2
0019-9567/04/$08.00+0 DOI: 10.1128/IAI.72.2.684-690.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Department of Microbiology and Immunology, Chandler Medical Center, University of Kentucky, Lexington, Kentucky 40536-0298,1 Foodborne and Diarrheal Diseases Branch, Division of Bacterial and Mycotic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, 303332
Received 3 March 2003/ Returned for modification 29 April 2003/ Accepted 28 October 2003
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CDT is produced by several different bacterial species, including Escherichia coli, Shigella spp., Campylobacter jejuni and related thermophilic campylobacters, Haemophilus ducreyi, Actinobacillus actinomycetemcomitans, and enterohepatic Helicobacter spp. (5, 18, 31, 35, 37). In all of these species, CDT activity is encoded by three adjacent genes, cdtA, cdtB, and cdtC, and the expression of all three genes is required for the production of active CDT (31, 34). CDT production by E. coli strains was first documented by Johnson and Lior (18), and the cdt genes from three different E. coli strains were subsequently cloned and sequenced (29, 30, 34). These sequence data indicated that there is heterogeneity present in the E. coli cdt genes. For example, the predicted amino acid sequences of CdtA, -B, and -C from E. coli strains 9142-88 and 6468/62 have 48, 61, and 42% identical and conserved amino acids, respectively (30, 34). The third strain sequenced, S5, is most closely related to strain 9142-88, with CdtA, CdtB, and CdtC proteins that have 93, 94, and 94% identical and conserved amino acids, respectively (29). These CDTs have been called type I (strain 6468/62), type II (9142-88), and type III (strain S5), based on the order of publication of their gene sequences (29).
In this report, we have examined cdt sequences from a variety of E. coli strains in order to determine if additional sequence variants exist and to look for the possible association of specific types of cdt sequence variants with different E. coli virulence markers. Furthermore, we have tested the ability of PCR methods to detect and distinguish between the CDT types produced by these E. coli strains. Overall, our results show that these CDT-producing strains can be divided into three groups based upon their complement of virulence-associated genes and their type of CDT. In addition, we show the existence of two new E. coli CDT sequences that have not been previously reported.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PCR. An initial multiplex PCR analysis to determine the presence of diarrheagenic E. coli virulence genes was carried out as described previously (38). This analysis detects the presence of the estI, eltI, stx1, stx2, eae, cnf1, Ehly, ipaH, uidA, and bfpA genes and the EAF plasmid. Additional PCR tests were done to analyze for the presence of cnf2, estII, and F17 fimbriae (2, 27, 28). eae subtypes were determined by PCR analysis (1, 10, 22, 33).
PCR detection of cdt genes began with the use of the degenerative primers VAT2 and WMI1, according to the method of Pickett et al. (31). These primers are based on two highly conserved regions in cdtB and will amplify, in E. coli strain 9142-88, a 510-nucleotide cdtB fragment encoding 170 amino acids (30, 31).
Additional PCR analysis of the cdt genes included use of the reactions described by Okuda et al. (25) for detection of sequences related to the type I CDT sequence found in E. coli strain 6468/62 (34) and the type II CDT sequence from strain 9142-88 (30), as well as the PCR described by Clark et al. (3) for the detection of type III CDT sequences, based on that found in E. coli strain S5 (29).
Our type I- and type II/III-specific PCR analysis of the cdtB gene described in this work used four sequence-specific primers. Two of these primers were based on the type I cdtB sequence, and two were based on the type II cdtB sequence (30, 34). The type I primers were type I-F, 5'-GCTTCTGCAACGTT(G,T)ACTGAG-3', and type I-R, 5'-AATCGCAAGATTTACTCTGTTAGCG-3', and the type II primers were type II-F, 5'-TCAACGGCTGTA(A,G)ATACAGG-3', and type II-R, 5'-CAGAGCAAGATTGACTCTTCCACCAAG-3'. These forward and reverse primers are based on amino acids 44 to 50 and 86 to 94 (Fig. 1), respectively. The forward primers were used in combination with the reverse primer WMI1; the reverse primers were used in combination with the forward primer VAT2. PCRs and cycle parameters were as described by Eyigor et al. (9), except that the annealing temperature was 47°C. All PCR primers were synthesized by Integrated DNA Technologies (Coralville, Iowa).
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Cloning and sequencing. Total DNA was isolated from E. coli strains using a QIAamp tissue kit (Qiagen Inc., Valencia, Calif.), after which the TOPO TA cloning kit (Invitrogen Corp., Carlsbad, Calif.) was used to amplify and clone a portion of the selected strain's cdtB gene. The degenerative sequences, VAT2 and WMI1, described above were used as primers in the amplification. At least two clones originating from each strain were sequenced (SeqWright, Houston, Tex.).
Nucleotide sequence accession numbers. The nucleotide sequences of the partial cdtB genes have been assigned GenBank accession numbers AY423896, AY423897, AY426340, and AY426341.
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RESULTS |
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Type-specific PCR for classification of cdt genes. Examination of the different partial cdtB sequences suggested that it would be possible to make PCR primers that were specific for either type I or for type II and III (II/III) cdtB sequences. We used four specific primers in combination with VAT2 or WMI1 to test whether we could use these primers in a screening PCR to determine if an E. coli strain likely had a type I or type II/III cdtB gene. We first tested the primers with several strains for which the sequence in the relevant region had already been determined. In every case, the type I primers, in combination with VAT2 or WMI1 (Table 2), amplified only sequences of the expected sizes from type I strains. Figure 2 shows the results for two known type I strains, 3061-99 and 3015-99. A PCR product was produced with only one of the two primer sets for each strain. For example, type I strain 3061-99 yielded a product with the type I primers (lane 2), but not with the type II/III primer set (lane 8). Similarly, the type II/III primers only amplified sequences of the expected sizes from known type II or type III strains (Table 1; Fig. 2, lane 11; and data not shown). We then tested the primer sets on eight additional CDT producers whose cdtB gene had not been sequenced. Of these strains, two produced a product with the type I primers (Table 2; Fig. 2, lanes 3 and 7) and the remaining six strains produced a PCR product only when tested with the type II/III primers (Table 2 and Fig. 2, lane 10, for strain 3084-98). All of the strains acted in a consistent and reproducible manner, suggesting that these type-specific primers can be successfully used to differentiate type I from type II and type III CDT-producing strains. These primers cannot distinguish between type II and type III sequences.
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Correlation of strain characteristics and CDT type. Analysis of the strains' known virulence factors and serotypes in relation to CDT type revealed some interesting associations (Table 1). Of the eight strains identified as encoding type I CDT, five were serogroup O2 and, of these, three were O2 nonmotile strains. Three of the strains, H1, H19, and 2P6, were initially identified as CDT producers in a study by Johnson and Stell (17) of E. coli strains associated with urosepsis, and certain O2 serotypes are well documented to be relatively common causes of this type of extraintestinal infection. However, the other two O2 strains with a type I CDT were also not stool isolates; 3008-99 was isolated from the urine of a 77-year-old female, and strain 3015-99 was isolated from the blood of a 40-year-old female. The other three type I strains, 3180-98, 3061-99, and 3142-98, were all isolated from human stools but, in common with the O2 serogroup type I strains, they did not encode Shiga toxin, enterohemolysin, or intimin. However, only the O2 serogroup type I strains possessed pap genes as assessed by PCR (reference 17 and data not shown). These results do not show that all CDT type I strains form a distinct clone, but they do indicate that at least a subset of type I CDT-producing strains appear to be associated with E. coli extraintestinal infections and that serogroup O2 strains that produce CDT likely possess type I CDT genes. In general, type I CDT-producing strains share the distinction of not having any of several known E. coli diarrheagenic-associated virulence markers (Table 1).
The type II CDT-producing strains varied from the type III strains based both on their cdtB fragment sequence and upon their complement of known virulence markers. There were three type III strains that encoded Stx1 and CNF2 but not intimin (Table 1; Fig. 3D); these are the same three strains that produced a product with the type III primers based on the cdt sequences in strain S5 (Fig. 3C). These strains appear to be similar to some of the bovine strains described by De Rycke et al. (7) in that they encode CDT type III and CNF2 but not F17 fimbriae (data not shown), although two of these three strains were isolated from humans. In any case, these strains appear to define a group of similar CDT-producing strains that make a type III CDT and also produce CNF2 and Stx1. The other known type III strain, 3139-97, encoded Stx1, Stx2, and the enterohemolysin. This strain was not identified by the type III-specific primers of Clark et al. (3) and thus appears to be distinct from the already-mentioned group of type III strains.
The three sequence-verified type II strains encode intimin, but apparently they encode no known E. coli toxins other than CDT (Table 1). In addition, one of the nonsequenced type II/III strains was identified as type II on the basis of a type II-specific PCR. This strain, 3084-98, also possesses an eae gene but none of the other virulence genes tested for in this work. None of these eae-carrying type II CDT producers tested positive for the presence of the EAF plasmid and, of note, all were nonmotile. These four strains appear to constitute another group of strains with significant commonalities, although their pathogenic significance is presently unknown. However, one of these CDT type II strains, 3260-97, is one of five strains that were described previously by Janda et al. (15) as either unusual biotypes of E. coli or as representatives of a new Escherichia species. All five of these strains were isolated from humans with diarrheal disease, and all five produce CDT (data not shown).
In addition to the six type II or III strains for which partial cdtB sequence data were obtained, six additional type II or III strains are shown in Table 1. Two of these strains, 3084-98 and F5953-98, were identified as to their CDT type based on the type II- and III-specific PCR methods discussed above (3). The other four strains have not yet been conclusively identified as to their CDT type, other than that they belong to either type II or type III (Tables 1 and 2). Three of these strains encode Stx2, and one of these also encodes Stx1. Our results from the sequenced strains would suggest that these strains are likely type III strains. The fourth strain identified as carrying either type II or III cdt genes, 3014-99, encoded neither intimin nor any toxins other than CDT in repeated PCR tests.
RAPD analysis. In an effort to learn more about the possible relatedness of strain 3014-99 to selected representatives of the different CDT types, RAPD analysis was performed. Each strain listed in Table 1 as well as some known siblings of selected strains were tested. These experiments are continuing, but our results, some of which are shown in Fig. 4, indicated that strain 3014-99, which has a type II/III partial cdtB sequence based on type-specific PCR, had a primer 2 RAPD pattern identical to that of several type I strains, including 3061-99, 3008-99, and 3015-99 (Fig. 4, lanes 5, 6, 7, and 8). The 3014-99 patterns achieved with primers 1 and 3 also appeared to be closely similar to that of the type I group, including strain 3008-99 (data not shown).
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DISCUSSION |
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Overall, our isolates included several strains with CdtB partial sequences that were identical to the prototype type I and type III sequences, but two strains possessed CdtB sequences closely related, but not identical, to the type II and III sequences of both S5 and 9142-88. We have chosen not to assign these strains separate types (e.g., types IV and V) but rather to call them variants of the type II/III group, since their cdt genes have not been sequenced in their entirety and because it is not known if the Cdt proteins produced by these strains differ significantly in their antigenic specificities or in any other way.
It is possible that there are other substantially different types of E. coli cdt sequences, but the use of the degenerative primers VAT2 and WMI1 for our initial screen for the presence of cdt sequences was chosen in order to detect as many variants as possible. These primers are derived from regions of CdtB that are very highly conserved in all CdtBs and have been used to detect cdt sequences in several different genera (e.g., references 6 and 26).
While sequence data suggested two general groups of CDT producers, analysis of the strains' complements of virulence markers suggested that there were really at least three basic types of CDT producers. The type I CDT-producing strains analyzed here produced no other toxins besides CDT, with the exception of one strain that encoded cytotoxic necrotizing factor type 1 (CNF1). Several of the type I CDT strains were serogroup O2 and all of these possessed pap genes, indicating that these strains probably belong to the well-studied O2 group of extraintestinal (uropathogenic) strains. The other type I strains did not possess pap genes, and more study will be needed to determine whether these strains have a similar pathogenic potential.
Type II/III strains seemed to fall into two classes: those that make intimin but not any known toxins other than CDT, and those that make one or more Shiga toxins. Interestingly, the latter group included the three strains 3032-96, F6450-99, and 3139-97 whose partial CdtB sequences were identical to that of S5. PCR tests for other virulence markers indicated that the unsequenced strains F5953-98, 3148-97, F5971-99, and F6448-99 also belong to this group of strains that encode Shiga toxin. Three of these seven Shiga toxin-producing strains also encoded CNF2, suggesting a relationship with the CNF2 producers profiled by De Rycke et al. (7). However, only one of our CNF2-producing strains, F5953-98, was a bovine isolate, suggesting that at least some type III CDT-producing, CNF2-producing E. coli strains can affect humans.
On the other hand, the strains that encode intimin and CDT included 9142-88, the prototype type II strain, and the two strains, 3102-97 and 3260-97, that possessed the new variant CdtB sequences shown here (Fig. 1). In addition, strain 3084-98 can be included in this group since it encodes intimin and CDT, and it was identified as a type II strain by type-specific PCR (Fig. 3B). These strains, which we are calling type II strains, appear to form a group of CDT producers that possess similar virulence markers and that have related, but not necessarily identical, cdt sequences. The pathogenic potential of this group is unclear, but their identification as a subset of CDT producers should facilitate future studies aimed at determining CDT's role in E. coli disease.
On the genetic level, the variation seen in the type II strains' cdt sequences may reflect accumulated changes since acquisition of ancestral cdt genes, or it may mean that multiple transfers of closely related but nonidentical cdt sequences have occurred within this group of Eae-producing strains. The former seems a more likely hypothesis, given the similar complement of virulence-associated genes present in these strains. On the other hand, the cdt sequence identity found in the type III strains may argue for more recent acquisition of the cdt genes by these Shiga toxin-producing strains.
Strain 3014-99 presents an interesting exception to the general correlations described for the type I, II, and III strains. Based on PCR tests, this strain produces type II or III CDT, yet the overall genetic make-up of the strain, reflected in RAPD analysis, and also its lack of genes encoding either Shiga toxin or intimin appear to indicate that it is more closely related to type I CDT-producing E. coli than to the type II or III CDT producers. It will be of interest to further characterize both this strain as well as additional strains in related groups so that we can better understand the genetics, biology, and pathogenic potential of E. coli CDT-producing strains.
While preparing this report, a paper appeared by Clark et al. (3) which looked at the association of cdt genes with other virulence markers from selected groups of E. coli strains isolated from both animals and humans. Their results were somewhat similar to our correlations, but not identical. For example, they found that many type I-producing E. coli strains possessed no other known virulence markers, except that several type I strains also produced CNF1. They found that type III CDT producers were most often combined with either CNF2 or CNF2 and Stx1. These strains were generally derived from cattle, and thus the association with CNF2 and Stx1 is not surprising. Our greatest difference was in characterization of the type II and III strains. Our type II strains all contained eae genes, while only one of five of theirs did and it was in combination with cnf1. Also, two of our type III strains contained both stx1 and stx2 genes, while none of theirs did. In fact, only 2 of their 50 CDT producers contained an stx2 gene, while four out of seven of our type III strains had an stx2 gene. It should also be noted that their method of distinguishing the type of CDT differed from our methods. They used PCR primers based primarily on differences within the cdtA gene of the three prototype strains (18). In agreement with our studies of the cdtB gene from type III strains reported here, their type III sequences were identical to that of strain S5. (The cdt PCR products from type I and type II strains produced by these investigators based on sequences from strains 6468/62 and 9142-88, respectively, were not sequenced.) However, our data clearly indicate that sequences within the type II group can vary, and our use of their primers with our strains showed that, in fact, these primers did not detect or distinguish all CDT producers within the three CDT type groups. This in turn suggests that additional, as-yet-unsequenced variations exist and that use of degenerative primers for preliminary screening for CDT producers is warranted.
Our RAPD and sequence data support the notion that strains that produce a particular type of CDT may be more closely related to each other than to other E. coli CDT producers but that not all strains producing a particular type of CDT can be considered close relatives (e.g., type 1 strains 3008-99 and 3180-98). That is, strains that produce a particular type of CDT may comprise two or more clonal groups of E. coli pathogens. Our RAPD data also suggest that some of the E. coli CDT-producing strains, such as the type I strains 3008-99 and 3061-99, may be closely related to each other despite having different serotypes.
The results presented here are an attempt to begin defining what types of E. coli pathogens produce CDT. Our data indicate that a fairly defined subset of uropathogenic E. coli strains contain cdt genes, that additional extraintestinal isolates of uncertain clonal identity also can carry cdt genes, and that strains with at least some characteristics in common with Shiga toxin-producing E. coli and enteropathogenic strains can carry cdt genes. A recent report by Janka et al. (16) indicated that O157:H- strains and, rarely, O157:H7 strains may encode a type III-like CDT. In addition, Mainil et al. (24) recently described a variant of type I CDT which they termed type IV CDT and which was found in some CNF1-producing E. coli strains from a variety of sources. Additional combinations of E. coli virulence genes among CDT producers appear likely to exist given the variation seen here and that reported by others.
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ACKNOWLEDGMENTS |
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Expert technical assistance was provided by Evan Sowers (Georgia) and Daniel Cottle (Kentucky).
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FOOTNOTES |
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X174; 2 and 8, 3061-99; 3 and 9, 3142-98; 4 and 10, 3084-98; 5 and 11, 3260-97; 6 and 12, 3015-99; 7 and 13, 3008-99. Selected DNA fragment standard sizes, in base pairs, are shown to the right.
