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Keywords:

  • CTX element;
  • genetic arrangement;
  • PCR;
  • pulsed-field gel electrophoresis;
  • Southern blot analysis;
  • Vibrio cholerae

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The composition and gene arrangement of the CTX genetic element were compared in 36 Vibrio cholerae isolates obtained during 2004–2006 from Iran. Long-PCR amplification of the CTX genetic element, using primers targeting ig1 and attB2, revealed three PCR products of c. 6.9, 5.6 and 2.6 kb, respectively. Southern blot hybridisation revealed that 30%, 17% and 53% of the isolates had one, two and three copies of the zot gene, respectively. PCR analysis of internal regions showed that isolates with three copies of the CTX genetic element carried one complete (6.9 kb) and two truncated CTX elements (each of 5.6 kb). In contrast, isolates with one or two copies of CTX carried the complete 6.9-kb CTX element. Pulsed-field gel electrophoresis revealed two pulsotypes among the isolates, with 75% of the isolates belonging to pulsotype  2. The pulsotype  2 isolates had varying CTX genomic arrangements, whereas the pulsotype  1 isolates had a homogeneous CTX arrangement. Thus, variations in the content, arrangement and copy number of the CTX genetic element may occur in isolates belonging to the same clone.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cholera is an infectious disease that causes severe diarrhoea and is a major public health problem in developing countries [1–3]. Cholera toxin (CT) is the key virulence factor of Vibrio cholerae, and this toxin is encoded by the ctxAB operon, which resides in the genome of a filamentous bacteriophage (CTXφ) that specifically infects V. cholerae [3,4]. Multiple copies of the CTX prophage are arranged tandemly in El Tor strains of V. cholerae, but the number and arrangement of the CTX elements and their associated repetitive sequences can vary [5,6]. The V. cholerae CTX genetic element has been fully sequenced [7], and a single copy of the cholera toxin genes, ctxAB, located on chromosome 1 within the integrated genome of CTXφ, has been reported in V. cholerae El Tor strain N16961 [8].

It has been shown previously that the genome of CTXφ contains a 4.5-kb central core region comprising the ctxAB, zot, ace, orfU and cep genes. A central core with a repetitive sequence (RS) of 2.4 kb, designated RS2, constitutes the CTX genetic element [9,10]. The CTX genetic element can also be flanked by one or more copies of a 2.7-kb RS, designated RS1. The RS2 region, which is located upstream of the CTX central core, contains three open reading frames (ORFs) that are involved in the regulation (rstR), replication (rstA) and integration (rstB) of CTXφ, together with two intergenic sequences, ig1 and ig2, at the left and right ends, respectively of rstR [6,11]. The RS1 element, a satellite phage, is very similar to the RS2 region of CTXφ, but with an additional ORF (rstC) [12,13]. At the left of the CTXφ genetic element is a toxin-linked cryptic (TLC) element, and on the other side of CTXφ is a region encoding an RTX toxin (rtxA), together with its activator (rtxC) and transporter (rtxBD) genes [14].

The aim of the present study was to investigate the diversity and genetic arrangement of CTX elements in 36 V. cholerae isolates obtained during 2004–2006.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacteria

In total, 36 clinical isolates of V. cholerae O1 El Tor from outbreaks during 2004–2006 were studied. The isolates were identified by biochemical assays and confirmed by PCR amplification of the 16S–23S rRNA intergenic regions specific for V. cholerae [11]. Serotyping was performed using monoclonal O1 antiserum and monospecific Ogawa and Inaba antisera (Mast Diagnostics, Bootle, UK). V. cholerae ATCC 14035 was used as a positive control.

PCR assay

PCR was used to analyse the composition and arrangement of genes within the CTX core or RS regions, using primers specific for the ctx, zot and ace genes [13] (Roche, Mannheim, Germany). The following primers, targeting different internal genetic regions, were also used: ig1 (5′-ACATAAGCGACGTAGCGTG), rstC-R (5′-GCTCAGTCAAT GCCTTGAGTTG), rstA-R (5′-GCATAAGGAACCGACCAAGCAAGAT) [15], rstR-F (5′-GCACCATGATTTAAGATGCTC) [13], attB2-R (5′-ACTTTGGTGC ACACAATTGACG), attB1-F (5′-CGCAGCAGACGAACTCTATGTC) [1], attRS-F (5′-CCTTAGTGCGTATTATGT) and attRS-R (5′-ACATAATACGCACTA AGG) [9].

Long-PCR (L-PCR) assay

The Expand Long Template PCR System (Roche), using primers ig1-F (targeting the 3′-end of rstR) and attB2-R (targeting the intergenic region between CTX and RTX), was used for amplifications (Fig. 1). A nested PCR program was used, with the first run comprising 2 min at 94°C, followed by 10 cycles of 15 s at 95°C, 30 s at 59°C and 8 min at 68°C, followed by a second run comprising 20 cycles of 20 s at 95°C, 30 s at 61°C and 7 min at 68°C (increasing by 20 s each cycle) and a final extension for 7 min at 68°C. Amplified fragments were then subjected to restriction fragment-length polymorphism analysis using EcoRV, DraI, BglI and XbaI (Roche).

image

Figure 1.  Schematic arrangement of the structure of the CTX genetic element and the flanking regions of strain N16961. Open reading frames are shown as arrows. The black triangles represent CTXφ attachment sites (attRS). Solid bars indicate intergenic regions flanking rstR; patterned bars indicate the attB1 and attB2 regions.

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Southern blot hybridisation of toxin genes

DNA was extracted from cultures grown overnight in brain heart infusion using phenol/chloroform/isoamyl alcohol extraction. The DNA was then digested with PstI (Roche), which does not cut within zot or ctxAB. In the CTX genetic element, PstI has only one cut site inside orfU [2,7] (Fig. 1). The resulting fragments were separated by agarose gel electrophoresis and then transferred to positively charged nylon membranes (Roche). A DIG DNA Labelling and Detection Kit (Roche) was used to produce digoxigenin-labelled ctxA and zot gene probes. The membranes were prehybridised for 30 min in a hybridisation buffer containing 5× SSC (20× SSC is 3 M NaCl, 0.3 M sodium citrate), blocking reagent (Roche) 2% w/v, N-laurylsarcosine 0.1% w/v and SDS 0.02% w/v. The membranes were then hybridised overnight with freshly labelled and denatured gene probes, and then washed twice in 2× SSC and SDS 0.1% w/v for 5 min at room temperature, and twice in 0.5× SSC and SDS 0.1% w/v for 15 min at 68°C. DNA fragments that hybridised with the probes were detected using anti-digoxigenin antibody according to the manufacturer’s instructions.

Pulsed-field gel electrophoresis (PFGE)

PFGE was performed using a CHEF-DRIII apparatus (BioRad, Hercules, CA, USA) and the Pulse-Net protocol for subtyping V. cholerae [16]. Genomic DNA was digested with NotI (Roche) and the restriction fragments were separated in Seakem Gold agarose (Cambrex, Rockland, ME, USA) 1% w/v in 0.5× TBE buffer (1× TBE buffer is 89 mM Tris, 89 mM boric acid, 2 mM EDTA). Salmonella enterica serotype Branderup H9812 DNA plugs digested with XbaI were used as DNA molecular size markers.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

PCR and L-PCR assays

L-PCR amplification of the CTX element was performed for all 36 clinical V. cholerae isolates using the ig1-F and attB2-R primers. The attB2 primer is specific for the intergenic region that lies between the CTX and RTX gene clusters (Fig. 1). PCR using primers specific for CTXφ prophage genes yielded three different amplicons with molecular sizes of c. 6.9 kb (designated pattern 1), 5.6 kb (pattern 2) and 2.6 kb (pattern 3) (Fig. 2a) in 30%, 53% and 17% of the isolates, respectively. The 6.9-kb products were digested with EcoRV (Fig. 2b, lane 1), DraI (Fig. 2c, lane 2), BglI (Fig. 2d, lane 1) and XbaI (Fig. 2d, lane 3), and this revealed that there were no differences in the restriction fragments generated from the isolates in the present study and those generated from V. cholerae N16961 (results not shown), suggesting that no genomic rearrangements, i.e., deletions or insertions, had occurred in the CTX genetic elements carried by isolates with pattern 1.

image

Figure 2.  Representative amplified DNA products obtained by long-PCR (L-PCR) using ig1-F/attB2-R primers. (a) Lanes 3, 2 and 1 are L-PCR products of patterns 3, 2 and 1, respectively. (b) Lanes 1, 2 and 3 show EcoRV digests of the L-PCR products of the three patterns. (c) Lanes 1 and 2 show DraI digests of the L-PCR products of patterns 1 and 2, respectively. (d) Lanes 1 and 2 show BglI digests and lanes 3 and 4 show XbaI digests of the L-PCR products of patterns 1 and 2. M, molecular size markers.

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L-PCR restriction fragment-length polymorphism (RFLP) analysis of the 5.6-kb pattern 2 amplicon with any of the four restriction enzymes suggested that the missing 1.3-kb genomic segment had been deleted from within the CTX core region (Fig. 2b, lane 2). PCR analysis of internal regions using the attB2-R primer and each of the forward primers targeting the ace, zot and ctx genes yielded amplicons with the expected sizes of 1.7 kb (ace-F/attB2-R) and 1.4 kb (zot-F/attB2-R). No PCR products were generated using the ctx-F and attB2 primers, suggesting that loss of the ctx gene had occurred in the isolates with a 5.6-kb CTX element (truncated CTX element). At the same time, PCRs targeting each of the toxin genes in these isolates were positive for the ctx, zot and ace genes (complete CTX element), suggesting the presence of multiple copies of the CTX genetic element.

The 2.6-kb amplicon of pattern 3 was investigated using attB2-R with either the rstR-F or ctxA-F primers, which yielded amplicons of the expected sizes of 2.4 kb and 4.1 kb, respectively. Amplicons with these sizes could only be obtained if RS1 is positioned at the right junction of the complete CTX element. The 2.6-kb amplicon (pattern 3) was then digested with EcoRV. The anticipated restricted fragment sizes confirmed the presence of an intact RS1 (Fig. 2b, lane 3). As expected, no digestion was observed with the three other restriction enzymes because of an absence of restriction sites within the 2.6-kb amplicon.

All isolates were examined for the presence of RS1 to the left of the CTX element by using primers specific for distal attB1, an intergenic sequence in the TLC element near CTX left, and rstC, at the 3′-end of the RS1 cluster. The anticipated 2.7-kb amplicon was obtained when the attB1-F and rstC-R primers were used, suggesting that RS1 was inserted in the left junction of the complete CTX element; However, for some isolates, RS1 was found to be located at the right junction of the CTX element.

Southern blot hybridisation

PstI-digested chromosomal DNA of all isolates was hybridised with probes for the ctx and zot genes. In general, the number of copies of ctxAB in an intact CTXφ genome should be identical to that of zot [2]. Three hybridisation profiles were detected, with bands ranging in size from 4.2 kb to 8.3 kb (Fig. 3). Hybridisation profile A (Table 1) revealed that the CTX element in 30% of isolates resided on the 5.6-kb fragments, which suggested the presence of one copy of the CTX element (genomic arrangement 1; Fig. 4). Hybridisation profile C (17%) indicated that the CTX genetic elements resided on two fragments of 8.3 and 6.9 kb, which suggested the presence of two copies of the CTX element (genomic arrangement 3; Fig. 4). Hybridisation profile B (53% of the isolates) suggested that three copies of the CTX element were present, located on fragments of 4.2, 5.6 and 6.9 kb, respectively, when the zot probe was used (genomic arrangement 2; Fig. 4). Moreover, when the same isolates were hybridised with the ctx probe, only one band of 6.9 kb was detected, suggesting that the ctx gene was missing from two copies of the CTX genetic elements.

image

Figure 3.  Southern blot hybridisation patterns of the Vibrio cholerae isolates obtained using (a) ctx and (b) zot gene probes. Lanes: 1 and 2, profile 1; 3 and 4, profile 2; 5 and 6, profile 3.

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Table 1.   Summary of Southern blot hybridisation profiles obtained following hybridisation of the zot or ctx probes with PstI-digested genomic DNA
Hybridisation profileNumber of bands detected usingCTX copy number% of isolates
zot probectx probe
A11130
B31353
C22217
image

Figure 4.  Proposed schematic genomic organisation of the CTX elements. The three possible arrangements found in all Vibrio cholerae isolates are labelled as 1, 2 and 3. The vertical arrows indicate the site of digestion by PstI. PstI restriction sites were determined from the fully sequenced V. cholerae strain N16961 [7] using NEB cutter software. inline image, complete CTX genetic element; inline image, TLC element; inline image, RTX cluster; inline image, truncated CTX genetic element; inline image, RS1; inline image, ctx probe; inline image, zot probe.

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PFGE

PFGE following NotI digestion of chromosomal DNA subtyped the 36 V. cholerae isolates into two pulsotypes, designated 1 and 2, respectively (Fig. 5). All of the isolates obtained during 2004 belonged to pulsotype  1 (Fig. 5, lane 1). Isolates obtained during 2005 and 2006 belonged to pulsotype  2 (Fig. 5, lane 2). Pulsotype  1 was similar to pulsotype  2, but had one missing 97-kb band. The most common (30%) genetic arrangement combined pulsotype  2 with the presence of one copy of the CTX genetic element. V. cholerae isolates belonging to pulsotypes  1 or 2 that carried three copies of the CTX element constituted 25% and 28%, respectively, of the isolates examined. The remaining 17% of isolates contained two copies of the CTX element and belonged to pulsotype  2. No relationship was revealed between the PFGE patterns of isolates and the geographical location from which they were isolated. Table 2 summarises the characteristics and genotypical analysis of the V. cholorae isolates included in the study.

image

Figure 5.  Pulsed-field gel electrophoresis patterns of the isolates obtained following digestion with NotI. M, molecular size marker. All Vibrio cholerae isolates belonged to either pulsotype 1 (lane 1) or pulsotype 2 (lane 2). Pulsotype 2 shows an additional 97-kb band.

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Table 2.    Comparative analysis of 36 sporadic Vibrio cholerae isolates obtained during 2004–2006
Strain no. Year/locationPFGE patternLong-PCR amplicon size (kb) (ig1-F/attB2-R)Hybridisation profileCTX copy numberSerotype
  1. PFGE, pulsed-field gel electrophoresis.

 12004/Iran West15.6B3Ogawa
 22004/Iran West15.6B3Ogawa
 32004/Iran West15.6B3Ogawa
 42004/Iran West15.6B3Ogawa
 52004/Iran West15.6B3Ogawa
 62004/Iran West15.6B3Ogawa
 72004/Iran West15.6B3Ogawa
 82004/Iran West15.6B3Ogawa
 92004/Iran West15.6B3Ogawa
102005/Iran North27.0A1Inaba
112005/Iran North27.0A1Inaba
122005/Iran North27.0A1Inaba
132005/Iran North27.0A1Inaba
142005/Iran Central27.0A1Inaba
152005/Iran Central27.0A1Inaba
162005/Iran Central27.0A1Inaba
172005/Iran Central27.0A1Inaba
182005/Iran Central27.0A1Inaba
192005/Iran Central27.0A1Inaba
202005/Iran West27.0A1Inaba
212005/Iran West25.6B3Inaba
222005/Iran West22.6C2Inaba
232005/Iran West22.6C2Inaba
242005/Iran West22.6C2Inaba
252005/Iran West22.6C2Inaba
262005/Iran West22.6C2Inaba
272005/Iran West22.6C2Inaba
282006/Iran Central25.6B3Inaba
292006/Iran Central25.6B3Inaba
302006/Iran Central25.6B3Inaba
312006/Iran Central25.6B3Inaba
322006/Iran Central25.6B3Inaba
332006/Iran Central25.6B3Inaba
342006/Iran Central25.6B3Inaba
352006/Iran Central25.6B3Inaba
362006/Iran Central25.6B3Inaba

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The results obtained by Southern blot hybridisation, L-PCR RFLP and PCR analysis of internal regions enabled all the isolates to be grouped into one of three different CTX genomic arrangements. Furthermore, it was revealed that up to three copies of the CTX genetic element could be inserted between the TLC and RTX genetic regions; however, these CTX copies were of variable sizes. The results suggested that 30% of the V. cholerae isolates carried one complete copy of the CTX element, in which RS1 was located to the left of the CTX element, adjacent to the TLC element. This arrangement (CTX genomic arrangement 1; Fig. 4) had RS1 at the opposite side to the arrangement in V. cholerae strain N16961, which contains RS1 to the right of the CTX element, flanking the RTX gene cluster [7]. CTX genomic arrangement 1 was predominant (61%) among the V. cholerae isolates obtained during 2005 that belonged to pulsotype  2. None of the isolates from 2004 and 2006 had this arrangement, and V. cholerae strain N16961 has been shown to carry only one complete CTX element [7].

Among all the isolates examined, 53% carried a truncated copy of the CTX element, with the loss of a 1.3-kb genomic segment. Amplification of the internal regions revealed loss of the ctxAB gene in the truncated CTX element. The location of RS1 in these elements was also shown to be on the left side of the CTX element, adjacent to the TLC cluster (Fig. 4). This arrangement has also been reported by Nandi et al. [17], while other investigators have reported RS1 on both sides of the CTX element [18].

In total, 83% of the isolates yielded a 9.1-kb amplicon when the attB1-F and ctxA-R primers were used. This corresponded to the expected size of a complete CTX genetic element plus a RS1 gene segment. DNA amplification of some of these isolates with the attB2-F and ig1-R primers yielded a 5.6-kb band, suggesting the presence of a truncated copy of the CTX element to the right. Combined, these data suggest the presence of both a complete and a truncated copy of the CTX element at the left end (near TLC) and the right end (near RTX), respectively, in CTX genomic arrangement 2. Probing of the isolates with a probe for the zot gene suggested the presence of another truncated CTX element (Fig. 4). This CTX genomic arrangement was detected throughout the 3-year period of the study, thereby demonstrating its stability. CTX genomic arrangement 2 was in agreement with previous findings that ctxAB is absent in truncated CTX elements found in V. cholerae O1 and O139 serotypes [19–21].

In contrast to CTX genomic arrangements 1 and 2, RS1 was located to the right, next to the RTX element, in CTX genomic arrangement 3. Using the zot-F and attB2-R primers, a 5.4-kb PCR product was obtained, which is the expected size when the amplified product includes the RS1 segment and the genes following the zot gene. These data indicate that RS1 in these isolates was located to the right of the CTX element, adjacent to RTX. This arrangement is comparable to that in the strain studied by Bhadra et al. [22] and to that in V. cholerae strain N16961 [7]. Furthermore, the results revealed the presence of two complete CTX genetic elements in these isolates.

NotI-digested genomic DNA of all isolates revealed two similar pulsotypes. Other investigators have also reported that isolates with an identical PFGE pattern may have different CTX genomic arrangements [6,17]. Such variations have been attributed to the amplification, rearrangement and deletion of the CTX genetic element in isolates belonging to the same clone [2].

In conclusion, the presence in these isolates of different copy numbers of the CTX genetic element, with diverse content and genomic arrangements, suggests: (i) that V. cholerae has been infected on multiple occasions by different CTXφ phages, each carrying a CTX element with different genomic arrangement; (ii) that an infecting CTXφ phage might have carried multiple copies of the CTX elements, complete and truncated; and (iii) that when RS1 is present, it is always positioned next to a complete CTX genetic element.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The study was funded by the Pasteur Institute of Iran, grant numbers 170 and 312. Our special thanks go to M. Rahbar from the Iranian National Reference Laboratory for sending the isolates from 2006, and to Dr Soroush from the Center for Disease Control in Tehran for helping us to obtain the isolates from 2004 to 2005. The authors declare that they have no conflicting interests in relation to this work.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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