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1Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, University of Dublin, Dublin 2, Ireland 2Centre for DNA Fingerprinting and Diagnostics (CDFD), Nacharam, Hyderabad, 50 00076 India 3Owaisi Hospital and Research Centre, Deccan College of Medical Sciences, Kanchanbagh, Santoshnagar, Hyderabad, India 4Department of Gastroenterology, The Adelaide and Meath Hospital, Tallaght, Dublin 24, Ireland
Correspondence Cyril J. Smyth csmyth{at}tcd.ie
Received August 20, 2003
Accepted February 12, 2004
Sequence variations located at the signal sequence and mid-region within the vacA gene, the 3'-end of the cagA gene, the indel motifs at the 3'-end of the cag pathogenicity island and the regions upstream of the vacA and ribA genes were determined by PCR in 19 paired antral or antrum and corpus Helicobacter pylori isolates obtained at the same endoscopic session, and three antral pairs taken sequentially. Random amplification of polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (FAFLP)-PCR fingerprinting were applied to these paired clinical isolates. The FAFLP-PCR profiles generated were phylogenetically analysed. For the 22 paired isolates there were no differences within pairs at five of the genetic loci studied. However, six pairs of isolates (27 %), of which four were antrum and corpus pairs, showed differences in the numbers of repeats located at the 3'-end of the cagA gene. RAPD-PCR fingerprinting showed that 16 (73 %) pairs, nine of which were antrum and corpus pairs, possessed identical profiles, while six (27 %) displayed distinctly different profiles, indicating mixed infections. Three of the six pairs showing differences at the 3'-end of the cagA gene yielded identical RAPD-PCR fingerprints. FAFLP-PCR fingerprinting and phylogenetic analysis revealed that all 16 pairs that displayed identical RAPD-PCR profiles had highly similar, but not identical, fingerprints, demonstrating that these pairs were ancestrally related but had undergone minor genomic alterations. Two antrum and corpus pairs of isolates, within the latter group, were isolates obtained from two siblings from the same family. This analysis demonstrated that each sibling was colonized by ancestrally related strains that exhibited differences in vacA genotype characteristics.
Present Address: Department of Genetics, University of North Carolina, Chapel Hill, NC 27599-7264, USA. Abbreviation: FAFLP, fluorescent amplified fragment length polymorphism.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The occurrence of spontaneous mutations after serial passage in gnotobiotic piglets indicates that H. pylori can undergo host-specific adaptation (Akopyants et al., 1995). This suggests that a founder strain of H. pylori may need to alter its genome to adapt to a new environment. This theory was confirmed in a study where RAPD analysis showed that genetic rearrangements arise from a founder strain during chronic infection (Kersulyte et al., 1999). In addition, various DNA fingerprinting methods applied to two or more H. pylori isolates taken from the same patient have shown that the fingerprint profiles of such strains are highly similar, with minor band differences (Marshall et al., 1998; Kuipers et al., 2000). This implies that two or more isolates recovered from one patient may share an ancestral relationship with a founder strain but have undergone independent genomic alterations. This phenomenon has been termed microevolution (Marshall et al., 1995, 1998).
Fluorescent amplified fragment length polymorphism (FAFLP) is a genotyping technique that has been successfully used to investigate outbreaks of infection by Pseudomonas aeruginosa (Ahmed et al., 2002), Streptococcus pyogenes (Desai et al., 1998), Staphylococcus aureus (Grady et al., 2000), Neisseria meningitidis (Goulding et al., 2000), Vibrio cholerae (Thompson et al., 2003) and Mycobacterium tuberculosis (Ahmed et al., 2003). FAFLP analysis generates specific profiles for each strain under examination. Recently, FAFLP analysis has been applied to H. pylori isolates (Ahmed et al., 2001; Carroll et al., 2003). This suggests that this technique might detect minor genetic differences between paired H. pylori isolates, and hence be a useful tool for studying microevolution.
The genetic diversity associated with H. pylori has been shown to occur at many specific loci, e.g. at the signal sequence and mid-region within the vacA gene (Atherton et al., 1995), the 3'-end of the cagA gene (Yamaoka et al., 1998), the 3'-end of the cag pathogenicity island (cag PAI) (Kersulyte et al., 2000), the upstream regions of the vacA and ribA genes (Bereswill et al., 2000) and within the iceA and babA genes (Yamaoka et al., 1999; Pride et al., 2001). The sequence variants located within these regions assist in characterizing H. pylori clinical isolates, and may also help to differentiate between different strains infecting the same patient.
The present study genotypically characterizes a collection of paired H. pylori isolates and demonstrates that microevolution can be observed at a high frequency within these isolates using the highly discriminatory FAFLP fingerprinting technique.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Isolation of genomic DNA.
Bacteria were grown as lawns for 3 days at 37 °C on CAB plates containing defibrinated horse blood and antibiotic supplements as above. The bacteria were harvested using sterile swabs and resuspended in 1 ml sterile distilled water. Total genomic DNA was isolated by a chloroform/phenol method as described previously (Wilson, 1995).
PCR.
PCR was performed on chromosomal DNA isolated from 3-day-old cultures. The sequences of the oligonucleotides used as primers and their corresponding PCR product sizes are listed in Table 2. The signal sequences and mid-regions of the vacA gene, the cagA gene, the 3'-end of the cagA gene, the indel motifs at the 3'-end of the cag PAI and the regions upstream of the vacA and ribA genes were amplified in separate reactions using Taq polymerase (Promega). The reactions were carried out in a final volume of 25 µl containing 1 µl DNA template (100 ng µl–1), 2 mM MgCl2, 40 pmol each primer, 2.5 U Taq polymerase, 80 µM each of dATP, dCTP, dGTP, dTTP in 10 mM Tris/HCl buffer (pH 8.3), 50 mM KCl and 0.001 % gelatin and overlaid with 25 µl mineral oil. PCR was performed in a PTC-100 thermocycler (MJ research) under the following conditions: 5 min at 95 °C, then 35 cycles of 1 min at 94 °C, 1 min at 50–52 °C (depending on primers used) and 1 min at 72 °C. This was followed by 10 min at 72 °C. The reaction products were then stored at 4 °C. The amplified products were separated on a 1 % agarose gel in 1x Tris/acetate/EDTA buffer (40 mM Tris/acetate, 1 mM EDTA, pH 8.0) containing 0.5 µg ethidium bromide ml–1. The DNA was then visualized under UV light. Representative PCR products for each allele or sequence variant were sequenced for confirmation of their identities, and sterile distilled water and DNA from Escherichia coli strain XL-1 Blue were used as negative controls for PCR.
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RAPD-PCR.
RAPD-PCR fingerprinting was carried out as described previously (Marshall et al., 1995) using primer P1 (Table 2).
FAFLP-PCR.
Restriction and ligation reactions were carried out in the same mixture. Total genomic DNA (10 ng) was heated to 95 °C for 5 min and then cooled at room temperature for 10 min. The denatured DNA was then added to a tube containing 1x T4 DNA ligase buffer (containing ATP), 0.05 M NaCl, 0.1 mg BSA ml–1, 50 U EcoRI, 10 U MseI, 10 U T4 DNA ligase and double-stranded adaptors for the EcoRI site (5'-CTCGTAGACTGCGTACC-3' and 3'-CTGACGCATGGT TAA-5') and the MseI site (5'-GACGATGAGTCCTGAG-3' and 3'-TACTCAGGACTCAT-5'). The restriction ligation mixture was incubated at 37 °C for 2 h. The digested and ligated products were diluted 20-fold. Of this product, 1.5 µl was used for preselective amplification using amplification core mix (Perkin Elmer) and pre-selective primers (5'-GTAGACTGCGTACCAATTC-3' and 5'-GACGATGAGTCCT GAGTAA-3') in a final volume of 10 µl. The PCR conditions were: 20 cycles of 94 °C for 20 s, 60 °C for 30 s and 72 °C for 2 min. The reaction products were then stored at 4 °C. The PCR product obtained was further diluted 20-fold and 1.5 µl of the diluted product was used for selective amplification in 10 µl with an EcoRI + A/T/G/C primer (5'-GTAGACTGCGTACCAATTC-A/T/G/C-3') with a fluorescent label (6FAM, HEX, TET or JOE) and an MseI + 0 primer (5'-GACGAT GAGTCCTGAGTAA-3'). PCR conditions for selective amplification were as described previously (Ahmed et al., 2002; Carroll et al., 2003).
FAFLP-PCR products with formamide loading dye (1.5 µl final volume) were loaded on to an ABI Prism 377 XL-96 DNA sequencer (PE Biosystems) along with an internal lane standard GS-500 Rox (PE Biosystems). Fragment separation was allowed to continue for 5 h on a 4 % (w/v) denaturing polyacrylamide gel. Fragments were detected and compiled by the ABI Data Collection software (PE Biosystems). A gel image was generated and each lane was scanned to make individual electropherograms. Fragment analysis was performed with the GENESCAN Analysis 3.1 package (PE Biosystems).
Genotypic analysis.
Based on the repertoire of amplified FAFLP-PCR products, different FAFLP profiles were identified as ‘amplitypes'. These amplitypes were colour-coded and overlaid to identify traces, fluorescence intensity (peak height), data points on the gel and the frequency of monomorphic bands. The GENESCAN data of individual isolates were exported to a Genotyper 2.5 (PE Biosystems) for digitization and genotyping.
Amplified products were sized in base pairs with respect to defined marker sizes (Ahmed et al., 2001, 2002, 2003). The presence or absence of amplicons was scored by a Genotyper macro (Ahmed et al., 2001, 2002, 2003). ‘Allele scores’ (presence or absence of amplicons) were converted into binary format (1, 0) and were used for establishing a similarity matrix needed for construction of a neighbour-joining network. A neighbour-joining tree was generated from the allele scores to delineate genetic similarities among the paired isolates.
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Sequences preceding the vacA and ribA genes
The intragenic sequence variants that precede the vacA and ribA genes were typed in all paired isolates. Again, no differences were detected in sequence variants preceding these genes within pairs. Of the pairs, 19 (86 %) were of type RibAP1, three (14 %) were of type RibAP3, 19 (86 %) were of type VacAP3 and three (14 %) were of type VacAP4. The combinations of these genotypes were RibAP1 VacAP3 (16, 72 %), RibAP1 VacAP4 (3, 14 %) and RibAP3 VacAP3 (3, 14 %). The two pairs of antrum and corpus isolates obtained from siblings displayed differences in the sequence variants preceding the vacA gene (577A and 577C, VacAP3; 9613A and 9613C, VacAP4) but not the ribA gene (RibAP1).
Insertion-deletion (indel) analysis
Indel motifs located at the right end of the cag PAI were examined. No differences were detected within pairs at this location in the genome. There were no differences in indel type (IV) between the two pairs of antrum and corpus isolates obtained from siblings of the same family. Of the pairs, 19 (86 %) yielded PCR products that could be used to determine which motif was located at the 3'-end of the cag PAI. Of these, nine (47 %) were of type II, eight (42 %) were of type IV and two (11 %) were of type Ia.
cagA genotyping
All paired isolates were found to possess the cagA gene. Further examination at the 3'-end of the cagA gene revealed that there was no variation in the number of repeat sequences within 15 pairs of isolates, of which six were antrum and corpus pairs. However, six pairs, of which four were antrum and corpus pairs, did display variation in the number of repeats in this region; the remaining pair (605A/605C) could not be typed within this region (Table 1). These differing pairs comprised isolates 45688A (one repeat)/45688C (two repeats), 15500A (one repeat)/15500C (three repeats), 87212A (two repeats)/87212C (one repeat), 79862A (one repeat)/79862C (two repeats), MI571 (one repeat)/MI575 (two repeats) and FR150 (one repeat)/FR151 (two repeats). The pairs of antrum and corpus isolates from siblings of the same family contained one repeat.
RAPD-PCR analysis
RAPD-PCR fingerprinting was carried out on the paired isolates to determine whether each pair represented isolates with identical (ancestrally related) or different (mixed infection) profiles. RAPD-PCR revealed that 16 (73 %) of the pairs, nine of which were antrum and corpus pairs, displayed identical fingerprint profiles, whereas six pairs (27 %), two of which were antrum and corpus pairs, displayed distinct fingerprint profiles. Within the latter six pairs, three displayed genetic identity at the six loci examined and might have been considered to be representative of single strains had this formed the basis for identity alone.
Of the six pairs of isolates showing variations in the number of repeats at the 3'-end of the cagA gene, three pairs (two antral pairs and one antrum and corpus pair) displayed different RAPD-PCR profiles (Fig. 1), while the other three pairs (antrum and corpus pairs) were ancestrally related on the basis of RAPD-PCR analysis. The two pairs of antrum and corpus isolates obtained from members of the same family that exhibited differences within the signal-sequence of, and sequence variants preceding, the vacA gene exhibited similar RAPD-PCR profiles for each isolate.
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FAFLP-PCR genotyping
FAFLP profiles were generated for the 22 paired H. pylori isolates to confirm earlier observations from RAPD-PCR fingerprinting and to determine whether minor genetic differences could be detected between paired isolates displaying identical RAPD-PCR profiles, as FAFLP-PCR is a more sensitive fingerprinting technique. Due to the fact that H. pylori isolates are genetically diverse (Go et al., 1996), if the FAFLP profiles of two isolates from the same individual displayed highly similar fingerprints they were accepted as being ancestrally related.
Visual analysis of the FAFLP-PCR fingerprints clearly indicated that some pairs showed nearly identical and others different profiles (Fig. 2). However, visual inspection is not enough to determine minor genetic differences between pairs.
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Three different electropherograms were generated for each of the 22 paired isolates, for each of the three selective primer pairs used (MseI + 0/EcoRI + A, + G or + C). The T selective primer (MseI + 0/EcoRI + T) did not generate fingerprints. The GENESCAN FAFLP electropherograms exemplify the diversity found between paired isolates (Fig. 3). These electropherograms revealed three situations that can occur between paired isolates: (i) all peaks, which represent amplified fragments, are shared by the isolates in a pair (Fig. 3a); (ii) the peaks in one isolate of a pair are not shared by its corresponding isolate (Fig. 3b) and (iii) isolates within a pair share the majority of peaks, but there are a small number of peaks that occur in one isolate and not the other (Fig. 3c), such minor differences being sometimes picked up by one selective primer pair but not another (Fig. 3d). Situation (i) indicates that the two isolates represent the same strain, situation (ii) indicates a mixed infection with two different strains and situation (iii) indicates a pair of ancestrally related isolates that exhibit microevolution.
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Phylogenetic analysis
The electropherogram peaks for all three selective primers were converted to binary format depending on whether a peak was present (1) or absent (0). The binary data for each isolate were aligned and a neighbour-joining phylogenetic tree was generated (Fig. 4). This tree shows that, out of the 22 pairs of isolates, the 16 (73 %) pairs that were identical by RAPD-PCR were found to have an ancestral relationship and the six pairs that exhibited dissimilar RAPD-PCR profiles (27 %) were found to be unrelated (mixed infection). With the exception of isolates 577A/577C and 9613A/9613C, which were obtained from siblings and grouped together, each isolate of the remaining 14 related pairs grouped as pairs. However, minor variations in their fingerprint profiles were detected as indicated by the differing branch lengths between isolates within each pair (Table 1). These minor variations indicated the presence of microevolution.
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The antral isolates 577A and 9613A from siblings of the same family are closely related genomically, yet have distinct VacAP and s/m types (Table 1). Interestingly, the corpus lineages 577C and 9613C are more related to each other than to their corresponding antral isolates in these genetically related human hosts.
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DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Factors that may impact on adaptation are (i) pH levels within different gastric niches and their influence on the survival of H. pylori and its ability to grow under acidic conditions (Karita & Blaser, 1998; Bijlsma et al., 2000) (ii) differences in the distribution of mucin glycoforms in the gastric mucosa (Nordman et al., 2002), (iii) differences in the viscosities of mucins and their influences on H+, HCO–3 and CO2 diffusion (Tanaka et al., 2002), (iv) differences in the biosynthesis of prostaglandins and hydroxyeicosatetraenoic acids in the regions of the stomach (Park et al., 2003), (v) differences in the binding of H. pylori to gastric mucins in a pH-dependent manner (Nordman et al., 1999) and (vi) differences in host genotype and physiology affecting niche characteristics (Akada et al., 2003). Alterations within the founder strain's DNA could potentially be selected for by these varying environmental factors found in different regions of the stomach. This would result in two populations colonizing different regions of the stomach but with highly similar genomes that display minor genetic differences.
All paired H. pylori isolates examined displayed identical allelic combinations at five different loci. To determine the relatedness of pairs of isolates RAPD-PCR was performed. This fingerprinting technique demonstrated that the majority of the pairs shared identical profiles. Of the six pairs that displayed different profiles, three pairs had differences in the numbers of 3'-cagA repeats and three had identical genotypes at six different loci (Table 1). This indicates that assessment of the relatedness of H. pylori strains residing in the same host by specific genotyping at multiple loci alone may not always be reliable (Kuipers et al., 2000; Owen & Xerry, 2003).
FAFLP-PCR analysis was in agreement with the findings of RAPD-PCR fingerprinting. However, the present study demonstrates that microevolved H. pylori isolates can be detected by applying the FAFLP-PCR fingerprinting technique. As this technique has a higher sensitivity, minor genetic differences between ancestrally related paired isolates were revealed that were not detected by RAPD-PCR typing. Although between some isolate pairs the same degree of diversity was obtained, e.g. 87212A/87212C (Table 1), for most pairs, one isolate exhibited two to six times the diversity of its paired isolate and, in one case (FR207/FR208), one isolate displayed over ten times the diversity of its corresponding isolate.
It is interesting to observe that two out of the three sequential paired isolates analysed in this study were found to be unrelated (MI571/MI575 and MI517/MI566). Antibiotic administration may have cleared the original H. pylori infection and allowed the establishment of a new one. H. pylori is known to be spread within families (Owen & Xerry, 2003). Incomplete eradication of H. pylori within either of these patients’ families could possibly account for the recrudescence of H. pylori infection with a new and unrelated strain via an infected family member.
The FAFLP-PCR findings with the paired antral and corpus isolates from siblings of one family are particularly intriguing. The signal-sequence of the vacA gene and its upstream region encompasses an approximately 400–500 bp segment of DNA, depending on the alleles and sequence variants present (Atherton et al., 1995; Bereswill et al., 2000). As it is unlikely that several insertion and deletion events occurred within one of these isolates altering its vacA genotype characteristics, these data strongly suggest that a recombination event occurred upstream of the vacA gene and within its 5'-end in one of these strains, leading to the conversion of a VacAP3 s1a genotype to a VacAP4 s2 genotype or vice versa. Previously evidence for the emergence of cag-recombinant strains during human infection has been presented by Kersulyte et al. (2000). Our findings represent the first report of a change in 5'-end vacA genotype characteristics during infection. Interestingly, a change in m-type, attributable to loss or gain of a 75 bp insert in the mid-region of the vacA gene, was reported between father and mother familial isolates that were otherwise identical in sequence type, allelic profile and deduced amino acid sequence for their housekeeping genes (Owen & Xerry, 2003). The stage of transmission at which the 5'-end vacA event might have occurred, i.e. parent to child, parent to parent or sibling to sibling, is unknown. After antral colonization, the 577A and 9613A populations appear to have adapted independently to the corpus regions of their individual hosts. This is consistent with the recent data of Akada et al. (2003) on how genetic divergence may affect tissue tropism.
The present study demonstrates that isolates with minor genetic differences detectable by DNA fingerprinting can arise during infection, yet it still remains uncertain as to where these alterations are within microevolved H. pylori isolates and what effect they have on the strain in relation to, for example, survival, acid tolerance, tropism or adherence. The minor differences observed in fingerprint profiles within paired isolates may be due to single base mutations, but equally may also be due to insertions, deletions or repeated sequences that commonly occur within H. pylori genomes (Taddei et al., 1997).
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ACKNOWLEDGEMENTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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REFERENCES |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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