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Final Report: Molecular Detection and Environmental Survey of Vegetative and Coccoid Helicobacter pylori
EPA Grant Number: R828037Title: Molecular Detection and Environmental Survey of Vegetative and Coccoid Helicobacter pylori
Investigators: Shahamat, Manouchehr , Alavi, Mohammad , Bradley, Brian , Gonzalez, Juan , Hind, John , Maeder, Dennis , Pavlick, Christine , Robb, Frank , Sowers, Kevin , Watts, Joy
Institution: University of Maryland Biotechnology Institute
EPA Project Officer: Nolt-Helms, Cynthia
Project Period: May 15, 2000 through May 14, 2003
Project Amount: $522,145
RFA: Drinking Water (1999)
Research Category: Drinking Water
Description:
The basic goals of this research proposal are to validate an accurate and sensitive molecular and/or immunodiagnostic method for rapid detection of H. pylori in both the culturable and in coccoid (i.e., VNC, viable, not culturable) states in environmental samples and examine the relationship between H. pylori and indicators of fecal pollution. This ability is crucial for preventing underestimation of the number of bacteria in samples, for developing epidemiological evidence, and for understanding the ecological and public health significance.
Objective:The objective of this research project was to develop a molecular method for rapid detection of Helicobacter pylori in the vegetative, culturable, and coccoid forms in environmental water. To achieve this goal, four laboratory approaches have been examined in the development of molecular detection of H. pylori from environmental water samples.
Laboratory Approaches
16S rRNA Hypervariable Flanking Region. Evaluation of the regions flanking the 16S and 23S rRNA genes of H. pylori with the aim of identifying specific sequences that could be used as molecular markers to identify H. pylori.
Proteomics. Analysis of the entire protein profile of the organism, or the proteome, for proteins specific to each morphological form and identify a unique protein expression (protein expression signature) for H. pylori under laboratory conditions. Identify those protein spots and select the most permanent one for making probes to be used as a molecular toll for identification of H. pylori.
Indirect Fluorescent Antibody. Application of monoclonal antibody specific to H. pylori to identify this bacterium in vegetative and in coccoid forms.
PCR Primers for glmM Amplification. Preparation and evaluation of highly specific DNA probes to identify DNA from H. pylori.
Approach:Phase 1: Immunological assay
- Produce monoclonal antibodies specific to the helical and coccoid form of H. pylori.
- Optimize a PCR based method using highly specific oligonucleotide probes for detection of vegetative as well as coccoid forms in environmental samples.
- Optimize and validate an immunodiagnostic protocol for rapid detection of the coccoid form in environmental samples.
- Examine the relationship between H. pylori and indicators of fecal contamination and initiate studies to identify the habitat of the organism in environmental samples.
After the four laboratory approaches were examined, two sets of primers were selected and used for polymerase chain reaction (PCR) amplification to identify H. pylori in cultures and water microcosms. H. pylori strains were selected from different geographical areas to examine the sensitivity and specificity of the primers. A number of strains belonging to other species of Helicobacter and bacterial genera also were tested to determine the specificity of the methods.
The first sets of PCR-based primers targeted the phosphoglucosamine mutase (glmM) gene and the second targeted the hypervariable region upstream of the 16S rRNA gene of H. pylori.
PCR Primers for glmM Amplification
Probes that specifically hybridize H. pylori genes encoding 16S rDNA glmM based on unique conserved consensus sequences for clinical strains were tested for sensitivity to several H. pylori strains isolated from biopsy samples. The PCR had a sensitivity of 0.1 picogram of H. pylori DNA. DNA was extracted from helical and coccoid forms of strains in spiked water. Other Helicobacter spp. including H. mustelae, H. felis, H. pametensis, and H. nemestinae also were tested. Two strains (H. mustelae and H. nemestrinae) were positive and the other two were negative with the glmM primers. Four strains of Campylobacter jejuni and 18 strains of other microbial species including four strains of C. jejuni, three strains of Ralstonia (27512, 27511, and 49129), three strains of Escherichia 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. aeru ginosa and P. putida), two species of Aeromonas (A. caviae and A. jandaie), one strain of Bacillus subtilis, one strain of Salmonella (S. dysenteriae), one strain of Shigella (Sh. typhimurium), one strain of Methanocaldococcus (M. jannaschii), and one strain of Acinetobacter tested negative with the glmM primers (see Figure 1). H. pylori also tested positive from a laboratory-prepared cocktail that included two strains of E. coli, Vibrio spp., Bacillus spp., Salmonella, and Shigella suspended in filtered river water and incubated at 15oC until use.
16S rRNA Hypervariable Flanking Region
Two primers were designed to amplify a unique hypervariable region upstream of the 16S rRNA gene of H. pylori. We have sequenced and compared this H. pylori conserved region from a number of H. pylori strains. The length of this region ranges from 992 to 1,548 base pairs depending on H. pylori strains. These primers were tested with DNA extracted from H. pylori, other Helicobacter spp., and a number of other species of bacteria. A positive PCR signal was detected for all 32 strains of H. pylori tested. In contrast, other Helicobacter spp. (H. mustelae, H. felis, H. fennellia strains 35684 and 35683, H. muridarum, H. canis, H. hepaticus, H. pullorum, H. bilis, H. bizzozeronii, H. cholecystus, Arcobacter butzleri, H. rappini, H. heilmannii, and H. trogontum) 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, a local hospital, and local farms), three strains of Ralstonia (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 species of Aeromonas (A. caviae and A. jandaie), one strain of B. subtilis, one strain of B. stearothermophilus, one strain of Salmonella, one strain of Shigella, and one strain of Acinetobacter. The selectivity of the assay was confirmed further by detection of H. pylori in a laboratory-prepared cocktail spiked with H. pylori cells as well as from a mixture of a variety of bacterial species including E. coli strains, Vibrio spp., Bacillus spp., Salmonella spp.,and Shigella spp.
The sensitivity of primers specific for the 16S rRNA hypervariable flanking region for the detection of H. pylori DNA was tested with serial dilutions of genomic DNA extracted from H. pylori type strain 11637. The PCR had a sensitivity of 2 picograms of H. pylori DNA as described by the method used in this experiment.
Tests performed with both primers using artificial mixtures of different bacterial species in well water and in river water gave only positive amplifications when Helicobacter spp. were present in the cocktails. In a series of experiments, the probes were applied directly to the water samples without DNA extraction, and H. pylori was identified when it was present in the sample. We were able to detect H. pylori for a 10 -year -old water microcosm directly without DNA extraction.
Conclusions:Currently, there are more than 20 Helicobacter spp., and more are being identified. Some of these non-pylori species can be present in clinical samples and others colonize animals. This diversity of Helicobacter creates a challenge for detecting H. pylori from environmental waters. The aim of this study was to develop a specific method for rapid detection of H. pylori in helical and in coccoid forms and to be able to selectively identify H. pylori in mixed populations of Helicobacter spp. and other bacterial spp.
In this study, two sets of PCR-based primers were evaluated to identify H. pylori from cultures and environmental water. The targets were the glmM gene and a unique sequence flanking a 16S rRNA gene sequence in H. pylori.
Experimental testing of a PCR primer pair resulted in a high specificity for detecting H. pylori strains. PCR amplification targeting this hypervariable region upstream of the 16S rRNA gene of H. pylori showed positive results for every H. pylori strain and negative for every species of Helicobacter (non-pylori species) and other bacterial species tested. In addition, the proposed PCR detection strategy for H. pylori using the 16S rRNA hypervariable region complements the results obtained by glmM-specific priming PCR amplification. Whereas the use of glmM-specific primers allows the detection of the Helicobacter genus (both H. pylori and some other species of the genus), PCR amplifications based on the proposed primers specific for the 16S rRNA flanking region of H. pylori discriminate the non-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 strain -detecting method because these sequences are highly specific for each H. pylori strain. For this purpose, more than 25 strains of H. pylori, other Helicobacter spp., and Campylobacter were sequenced in our laboratory, and these sequences have been deposited in the GenBank DNA database (accession n umbers AY505018 to AY505045), and they are available for homology searches using BLAST or similar algorithms in future applications and rapid identification methods. Both methods can complement each other in confirming results, which can be highly valuable in deciding the presence or absence of detectable levels of H. pylori in clinical specimens and environmental samples.
The specificity and sensitivity of these primers, particularly the 16S rRNA flanking region primers, will significantly help to detect accurately and rapidly H. pylori in environmental waters including drinking water, well water, and river water. By using these two PCR methods, one can detect Helicobacter spp. with the glmM probes and identify H. pylori using the specific primers targeting the 16S rRNA hypervariable flanking region proposed in this study. Recent published data indicate that H. pylori can be detected in different waters. Culturing techniques are not an option for most of these samples because of conversion of the bacterium to an unculturable coccoid form (see Figure 2). Application of our combination PCR-based approach allows same -day detection of H. pylori in cultures and in environmental water samples. Another advantage to our method is that only a small volume of the samples is needed. The molecular probes also can be used to detect H. pylori directly without DNA extraction.
Figure 1. Representative Results for PCR Products Generated From a Wide Spectrum of Bacterial Strains Using Primers for the H. pylori glmM Gene.
Figure 2. Nonculturable H. pylori in a Water Microcosm. Most of the cells converted to coccoid forms. A few horseshoe-shaped rods also are present.
Expected Results:We propose to: (1) produce a monoclonal antibody specific to H. pylori and develop an immunodiagnostic method for rapid detection of the bacterium in coccoid form; (2) develop highly specific PCR-based method for detection of H. pylori in the vegetative and coccoid forms in clinical and environmental samples; and (3) develop an oligonucleotide probe for detection of H. pylori in environmental samples, especially in coccoid form.
Journal Articles on this Report: 1 Displayed | Download in RIS Format
| Other project views: | All 7 publications | 1 publications in selected types | All 1 journal articles |
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Shahamat M, Alavi M, Watts J, Gonzalez JM, Sowers KR, Maeder DW, Robb FT. Development of two PCR-based techniques for detecting helical and coccoid forms of Helicobacter pylori. Journal of Clinical Microbiology 2004;42(8):3613-3619. |
R828037 (2001) R828037 (Final) |
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Helicobacter pylori, vegetative and coccoid forms, evaluation, molecular detection, proteomics, monitoring, oligonucleotide, public health,
, Water, Scientific Discipline, RFA, Drinking Water, Health Risk Assessment, Environmental Microbiology, Environmental Chemistry, bacteria, public health, treatment, exposure and effects, olgonucleotide, molecular detection, monitoring, detection, coccoid heliobacter pylori, environmental survey, fecal contamination, immunological assay, exposure, microbial contaminationProgress and Final Reports:
2001 Progress Report
Original Abstract