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The primary mode of transmission of Helicobacter pylori, a human pathogen carried by more than half the population worldwide, is still unresolved. Some epidemiological data suggest water as a possible transmission route. H. pylori in the environment transforms into a nonculturable, coccoid form, which frequently results in the failure to detect this bacterium in environmental samples by conventional culture techniques. To overcome limitations associated with culturing, molecular approaches based on DNA amplification by PCR have been developed and used for the detection of H. pylori in clinical and environmental samples. Our results showed the glmM gene as the most promising target for detection of H. pylori by PCR amplification. Under optimal amplification conditions, glmM-specific primers generated PCR-amplified products that were specific for H. pylori and some other Helicobacter species. Genome sequence analysis revealed the existence of a conserved region linked to a hypervariable region upstream of the 16S rRNA gene of H. pylori. Selective PCR primer sets targeting this sequence were evaluated for the specific detection of H. pylori. One primer set, Cluster2 and B1J99, were shown to be highly specific for H. pylori strains and did not produce any PCR products when other Helicobacter species and other bacterial species were analyzed. In tests with 32 strains of H. pylori, 6 strains of other Helicobacter species, 8 strains of Campylobacter jejuni, and 21 strains belonging to different genera, the primers for glmM were selective for the Helicobacter genus and the primers containing the region flanking the 16S rRNA gene were selective for H. pylori species only. The combination of two sensitive PCR-based methods, one targeting the glmM gene and the other targeting a hypervariable flanking region upstream of the 16S rRNA gene, are complementary to each other. Whereas the glmM-specific primers provide a rapid, sensitive presumptive assay for the presence of H. pylori and closely related Helicobacter spp., the primers for sequences flanking the 16S rRNA gene can confirm the presence of H. pylori and locate the potential source of this bacterium.
Helicobacter pylori is a gram-negative microaerophilic bacterium that infects human gastric epithelial cell surfaces and the overlying gastric mucin, which is a highly specialized niche. This bacterium is carried by more than half of the human population worldwide and is an important pathogen. It is the major cause of peptic ulcers and a contributor to other illnesses, ranging from childhood malnutrition to gastric cancer, and is known to be an agent causing susceptibility to other food- and waterborne pathogens (7, 10, 11, 39, 46).
Infection with H. pylori occurs worldwide, but the geographical prevalence varies greatly (41). Higher infection rates occur in developing countries, where 80% of middle-aged adults are infected compared with infection rates of only 20 to 50% in developed countries (16, 22, 42). Worldwide, the occurrence of H. pylori infection increases with age, and specific ethnic groups are also at greater risk (12, 19, 27, 31).
There is evidence that countries with poor sanitation have greater infection rates, which suggests a common water source as the reservoir rather than person-to-person transmission (1). There is a strong correlation between the consumption of municipal water and H. pylori infection in some countries (42). Recent data suggest that the bacterium is present in river water and drinking water (23, 24). However, the exact mode of H. pylori transmission remains largely unknown, as proof of a waterborne or food-borne route of transmission for H. pylori requires its detection from environmental reservoirs, foodstuffs, and water contaminated with human fecal material.
It has been observed that H. pylori cells can transform in vitro from a culturable spiral-shaped form to an as-yet nonculturable coccoid form. This change in morphology is accompanied by physiological changes that make it very difficult to recover the bacterium by routine culturing methods. These changes have been monitored in water microcosms in our laboratory (30, 38) and by other researchers (20, 45).
To overcome this problem, a variety of culture-independent methods have been tested for the detection of H. pylori in clinical samples (7, 9, 25, 36). PCR-based methods have also been used to detect H. pylori from environmental samples (1, 5, 15, 37). Most of these PCR methods target the 16S rRNA gene, random chromosome sequences, the 26-kDa species-specific antigen gene, the urease A (ureA) gene, and the urease C (ureC) gene (5, 28). Among these PCR methods, PCR-based detection of the ureC gene appears to be the most promising for detection of H. pylori (28). The ureC gene, renamed glmM by De Reuse et al. in 1997 (13), encodes phosphoglucosamine mutase, an enzyme catalyzing the interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate, which is subsequently transformed into N-acetylglucosamine (13). Although PCR amplification of glmM can detect H. pylori, we determined that it also detected a number of other Helicobacter species. Therefore, a complementary technique was needed to discriminate H. pylori specifically from other Helicobacter species.
Analysis of the complete genomic sequence from two strains of H. pylori, strains J99 and 26695 (2, 44) (GenBank accession numbers AE001439 and AE000511, respectively), has revealed that the 16S rRNA and 23S rRNA genes are not contiguous in the H. pylori chromosome. In all other species analyzed, the 16S and 23S rRNA genes are concatenated in their genome sequence. The regions flanking the 16S and 23S rRNA genes of H. pylori were examined with the aim of identifying specific sequences that could be used as molecular markers to identify H. pylori in environmental samples.
The goal of the research described herein was to design and validate an accurate and sensitive molecular method for rapid detection of both the helical and coccoid forms of H. pylori from culture, clinical, and environmental samples.
The H. pylori strains and other bacterial strains from diverse genera used to establish assay specificity are listed in Table Table1.1. Thirty-two strains of H. pylori were obtained from the National Collection of Type Cultures (Public Health Laboratory, London, United Kingdom), American Type Culture Collection, and our own collection, which included isolates from patients in local hospitals. Six strains of other species of Helicobacter, 8 strains of Campylobacter jejuni, and 21 strains belonging to different bacterial genera were included in this study (Table (Table11).
Growth conditions and media for all strains used in this study are shown in Table Table2.2. The cultures were sequentially transferred twice prior to testing. To induce the coccoid form of H. pylori, a freshly prepared suspension of H. pylori cells was inoculated into a series of prewashed 2-liter flasks, with each flask containing 1 liter of sterile drinking water, well water, and river water. The initial concentration of the cells was 107 to 108 cells/ml. Flasks were incubated at 15 and 4°C. At established intervals, samples were removed aseptically from the microcosms, cultured on blood agar plates for viable count, and simultaneously counted using the acridine orange direct count method of Hobbie et al. (21) with some modifications. Morphological and viability changes in the cells were monitored. After 20 to 30 days, 99% of the cells in the water microcosms converted into the coccoid form (30, 38).
DNA was extracted from 1-ml aliquots of sample (from culture or water microcosms) with the Fast DNA SPIN kit (Bio 101, Vista, Calif.) per the manufacturer's instructions. Briefly, the samples were added to lysing reagents in a matrix tube that contained silica and ceramic beads of various sizes. The cells were lysed in a Bio 101 Savant FastPrep bead beater (Savant, Farmingdale, N.Y.) for 30 s at 5.5 m s−1 and then centrifuged for 30 min at 10,000 × g. The supernatant was transferred to a tube containing a protein-precipitating solution and centrifuged for 10 min at 10,000 × g. The supernatant was removed, combined with the DNA-binding matrix, and centrifuged through a spin filter. The filter was then washed with a salt-ethanol solution and centrifuged. After the filter was dry, the DNA on the spin filters was eluted in 100 μl of sterile water (Sigma) and then purified by electrophoresis on a 1.3% (wt/vol) low-melting-point agarose gel. Chromosomal DNA was excised from the gel and recovered with a Promega Wizard PCR prep kit (Promega, Madison, Wis.), according to the manufacturer's instructions.
Oligonucleotides that specifically hybridize H. pylori genes encoding phosphoglucosamine mutase (glmM) and targeting conserved consensus sequences from clinical strains (28) were used in PCR amplification reactions. The primers used in this study are presented in Table Table3.3. All PCR amplifications were performed using the GeneAmp PCR kit with Taq DNA polymerase (Perkin-Elmer, Inc.) in a PTC200 thermal cycler (MJ Research, Watertown, Mass.). The PCR conditions described by Lu et al. in 1999 (28) were further optimized as follows: an initial denaturation step of 2 min at 95°C, followed by 30 cycles, with 1 cycle consisting of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C. A final extension step of 5 min at 72°C concluded the reaction. For controls, the 16S rRNA gene sequence was amplified using universal primers.
The PCR primers Cluster2 and B1J99 (Table (Table3)3) target sequences that flank a hypervariable region upstream of a 16S rRNA gene in H. pylori. These primers were designed on the basis of information from the complete genome sequences of two H. pylori strains J99 and 26995 (GenBank accession numbers AE001439 and AE000511, respectively). A 16S rRNA gene sequence from H. pylori strain 26695 was used as a query using the BLAST algorithm (3) against Helicobacter genomic sequences to find the number and location of the 16S rRNA gene sequences in the two published H. pylori genomes. The graphical interfaces GeneMate and Cross (http://comb5-156.umbi.umd.edu/genemate/getseq.html) were used to visualize BLAST results. PCR was performed by a hot start Taq polymerase (Qiagen, Valencia, Calif.) in a final reaction volume of 50 μl with 200 μM deoxynucleoside triphosphate and 0.4 μM concentration of each primer using genomic DNA as the template. Amplification conditions used to amplify the hypervariable region flanking the 16S rRNA gene were as described above except that an annealing temperature of 60°C and longer extension steps (1 min at 72°C) were used.
To determine the sensitivities of intergenic spacer region (ISR) primers, DNA extracted from H. pylori reference strain 11637 was serially diluted, and 1 μl of each dilution was used in the PCR. The starting concentration of DNA as determined by absorbance at 260 nm was 157 ng/μl. Serial dilutions were made by mixing equal volumes of the DNA and ultrapure water in consecutive tubes, with the final dilution having an estimated concentration of 0.5 pg/μl.
The PCR amplicons from the region flanking the 16S rRNA gene were cloned into Escherichia coli by using a TA-PCR cloning kit (Invitrogen) according to the manufacturer's instructions. The recombinant plasmids were purified using a miniprep plasmid purification kit (Qiagen) and sequenced using an ABI 377 DNA sequencer (Applied Biosystems, Foster City, Calif.). The DNA fragment between the primers Cluster2 and B1J99 was sequenced using M13 reverse and T7 promoter sequencing primers and the additional primers Hp2R and Hp1F. The primers Hp2R and Hp1F were based on the sequences downstream of the Cluster2 and B1J99 sequences, respectively. The sequences were then aligned using the computer multiple-sequence-aligning program ClustalW (43).
Two sets of primers were used for PCR amplification to identify H. pylori in cultures and water microcosms.
A total of 26 strains of H. pylori were tested with the glmM primers, and PCR results were positive for all strains tested. DNA was extracted from helical and coccoid forms of strains 52185, 61, 33097, and 11637 in spiked water. Other Helicobacter spp., including H. mustelae, H. felis, H. pametensis, and H. nemestrinae, were also tested. Two species (H. mustelae and H. nemestrinae) were positive, and the other two were negative with the glmM primers. Four strains of C. jejuni and 18 strains of other microbial species, including three strains of Ralstonia picketti (27512, 27511, and 49129), three strains of E. coli (K-12, HU735 isolated from human blood, and O157-H7), two vibrio strains (Vibrio cholerae O1 and Vibrio vulnificus), two Pseudomonas species (Pseudomonas aeruginosa and Pseudomonas putida), two strains of Aeromonas hydrophila (1028 and 1728), two other species of Aeromonas (Aeromonas caviae and Aeromonas jandaei), one strain of Bacillus subtilis, one strain of Shigella (Shigella dysenteriae), one strain of Salmonella (Salmonella enterica serotype Typhimurium), one strain of Methanocaldococcus (M. jannaschii), and one strain of Acinetobacter tested negative with the glmM primers (Fig. (Fig.1).1). H. pylori also tested positive in a laboratory-prepared cocktail that included two strains of E. coli, Vibrio, Bacillus, Salmonella, and Shigella spp. suspended in filtered river water and incubated at 15°C. The PCR had a sensitivity of 0.1 pg of H. pylori DNA as previously reported (28).
Two primers were designed to amplify a unique hypervariable region upstream the 16S rRNA gene of H. pylori. These primers were tested with DNA extracted from H. pylori, other Helicobacter species, and a number of other species of bacteria (Table (Table1).1). A positive PCR signal was detected for all 32 strains of H. pylori tested. In contrast, H. mustelae, H. felis, H. pametensis, H. muridarum, H. canis, and H. nemestrinae tested negative with these PCR primers. The PCR primers also did not hybridize with DNA from 28 other strains including eight strains of C. jejuni (from American Type Culture Collection, local hospital and local farms) three strains of Ralstonia picketti (27512, 27511, and 49129), three strains of E. coli (K-12, HU735 isolated from human blood, and O157-H7), two strains of vibrios (V. cholerae O1 and V. vulnificus), two species of Pseudomonas (P. aeroginosa and P. putida), two strains of A. hydrophila (1028 and 1728), two other species of Aeromonas (A. caviae and A. jandaei), one strain of B. subtilis, one strain of B. stearothermophilus, one strain of Salmonella, one strain of Shigella, and one strain of Acinetobacter (Fig. (Fig.2).2). The selectivity of the assay was further confirmed by detection of H. pylori in a laboratory-prepared cocktail spiked with a mixture of H. pylori, Escherichia coli strains, Vibrio spp., Bacillus spp., Salmonella spp., and Shigella spp.
The sensitivities of primers specific for the hypervariable region of H. pylori DNA flanking the 16S rRNA gene were tested with serial dilutions of genomic DNA extracted from H. pylori type strain 11637. The lower limit of detection was 2 pg of H. pylori DNA as found by the method used in this experiment (Fig. (Fig.33).
At this time, there are more than 23 Helicobacter species (6, 14), and more species are being identified. Some of these non-H. pylori species are found in clinical samples, and others colonize animals (18). This diversity of Helicobacter species creates a challenge for the detection of H. pylori from environmental waters. The aim of this study was to develop a specific method for rapid detection of H. pylori and to be able to selectively identify H. pylori in mixed microbial communities.
The detection of H. pylori is critical due to the high prevalence of this species, its worldwide distribution, and the large number of individuals carrying this species (7, 10, 11, 16, 38, 41, 46). In addition, H. pylori cells have the ability to form resistant, coccoid forms. H. pylori cells in the coccoid stage cannot be cultured and can be detected only by culture-independent strategies (5, 38).
H. pylori-specific probes that target the 16S rRNA, urease (ureA), and phosphoglucosamine mutase (glmM) (4, 28) genes have been proposed. The 16S ribosomal DNA and ureA primers did not detect all of the Helicobacter spp. under the reaction conditions described (data not shown). Results with a wide spectrum of bacteria tested in this study showed that the primers for glmM were selective for Helicobacter spp. and did not amplify products from non-Helicobacter spp.
In order to determine whether the assay was selective for Helicobacter spp., DNA from other species of Helicobacter and the closely related genus Campylobacter was tested under the same conditions. Initial results showed that although the assay detected all Helicobacter species, Campylobacter species were also detected (data not shown). Reaction conditions were modified to develop a more stringent assay specific for Helicobacter spp. as described in Materials and Methods. After this optimization, results showed that only Helicobacter spp. were detected, and several environmental strains of the closely related genus Campylobacter no longer yielded amplification products (Fig. (Fig.1).1). The PCR-based method targeting glmM was previously proposed by Lu et al. in 1999 (28), but they did not report whether the PCR method was tested on any other Helicobacter species. Bickley et al. (4) found that this PCR test was specific to H. pylori and did not yield any products when tested on five other species of Helicobacter. We found that this PCR assay using the same primers yielded positive results for H. pylori and some other Helicobacter spp. tested.
To overcome this limitation, another set of PCR-based strategies was evaluated to identify H. pylori from cultures and water microcosms. The target for this PCR was a unique sequence flanking a 16S rRNA gene sequence in H. pylori.
The ISR between the 16S and the 23S rRNA genes has been the target for designing species- and even strain-specific molecular probes. This has been possible due to the high inter- and intraspecific variability (8, 17, 33, 34, 40). The advantage of this region is that the flanking 16S and 23S rRNA genes at the 5′ and 3′ ends of the ISR contain highly conserved sequences (i.e., the 16S and 23S rRNA genes). Thus, conserved primers can be used for the selective amplification of ISR from bacterial species. However, in H. pylori, a ribosomal 16S rRNA gene is not followed in the genome sequence by the 23S rRNA gene. The rRNA operon structure of H. pylori has an atypical bacterial rRNA operon structure, as 16S rRNA sequences are physically removed from the 23S and 5S rRNA sequences and positioned on different DNA strands. There are two sets of rRNA genes in the genomes of H. pylori strains. The 16S rRNA genes for strain J99 are located on the plus strand at positions 1188029 to 1189529 and 1463047 to 1464547, and the 16S rRNA genes for strain 26995 are located on the plus strand at positions 1207583 to 1209081 and 1511137 to 1512634. The associated primary transcript is synonymous with the minus strand. These regions lack the context of the typical hypervariable ISR of the rRNA operon found in other bacteria. The intervening region is poorly conserved. This pairing is specific for one of the copies of the 16S rRNA gene, and no suitably conserved region was identified for the other copy of the gene.
A nonstandard ISR is present in H. pylori. Nevertheless, a highly variable region, likely a remnant of its primitive ISR, is followed by a short conserved sequence which could be used for the design of a diagnostic probe for PCR amplification.
We designed a primer pair specific for H. pylori (Cluster2 and B199 primers) and evaluated its specificity in discriminating non-H. pylori species while uniquely identifying H. pylori. The first primer (B1J99) overlaps the 16S rRNA sequence and is synonymous with the primary transcript to avoid hybridization with 16S rRNA. The second (Cluster2) represents a sequence conserved between H. pylori J99 and H. pylori 26695, about 1,000 bases upstream on the opposite strand. We have sequenced and compared this conserved region from a number of H. pylori strains. The length of this region ranges from 992 to 1,548 bp, depending on the H. pylori strain.
Experimental testing of the PCR primer pair resulted in a high specificity for detecting H. pylori strains. PCR amplification targeting this hypervariable region showed positive results for every H. pylori strain tested and negative results for every Helicobacter species (non-H. pylori species) and other bacterial species tested. These results suggest that the proposed primer set resulted in a specific detection strategy for H. pylori from cultures, clinical, and environmental samples. In addition, the proposed PCR detection strategy for H. pylori using the 16S rRNA hypervariable region complements the results obtained by PCR amplification with glmM-specific primers. While the use of glmM-specific primers allows the detection of Helicobacter species (both H. pylori and other species of the genus), PCR amplifications based on the proposed primers specific for the region flanking the 16S rRNA gene of H. pylori discriminate the non-H. pylori species of this genus. Furthermore, sequence comparison using the proposed hypervariable region upstream of the 16S rRNA gene of H. pylori can be an easy way to distinguish H. pylori strains, since these sequences are highly specific for each H. pylori strain. For this purpose, these sequences have been deposited in the GenBank DNA database (accession no. AY505018 to AY505034, AY505037 to AY505043, and AY505045) and they are available for homology searches using BLAST (3) in future applications and rapid identification methods.
In addition, PCR amplification using the proposed primers allows detection not only of H. pylori but also of H. pylori strains, as the amplified product can be used to differentiate strains of this species. Review of the literature has not revealed other published studies that used PCR amplification targeting the proposed hypervariable region upstream of the 16S rRNA gene in H. pylori. The specificity and sensitivity of these primers, particularly the primers containing the region flanking the 16S rRNA gene, will significantly help to accurately and rapidly detect H. pylori in environmental waters, including drinking water, well water, and river water. This is the first report proposing the use of this DNA sequence for the rapid detection and identification of H. pylori in both helical and coccoid forms. Recent published data indicate that H. pylori can be detected in different water samples (5, 15, 20, 29, 32). Culturing techniques are not an option for most of these samples due to conversion of the bacterium to the nonculturable coccoid form. Although the PCR detection system does not distinguish viable from nonviable bacteria in any given sample, our combination PCR-based approach provides the effective presumptive assay for determining the presence of H. pylori in cultures and in environmental water samples on the same day. Since the coccoid form of H. pylori represents a persistent form which can exist in water for long periods of time, positive results obtained by using these probes could indicate the presence of such cells.
This research was funded in part by EPA's Science to Achieve Results (STAR) program grant R82803701. J.M.G. is supported by a contract from the Spanish Ministry of Science and Technology (Ramon y Cajal program).