Abstract
Background and Aim
H. pylori can survive long incubation periods under anaerobic conditions, and should be possible to isolate under anaerobic conditions. Our aim was to isolate H. pylori in anaerobic conditions, from gastric biopsies of H. pylori infected patients.
Methods
We enrolled 27 patients with bleeding (erosive) gastritis (mean age 36.3 years, 55.6% male) from Hanoi, Vietnam. H. pylori status was confirmed by qPCR.
Results
H. pylori were recovered under anaerobic and micro-aerobic conditions from gastric biopsies in 16 patients. Anaerobic conditions yielded significantly higher H. pylori recovery rates than micro-aerobic conditions (81.3% vs. 31.3%, p=0.01). H. pylori isolates were characterized by PCR for specific virulence markers and the genotypes were similar to those previously described in this region of the world.
Conclusions
H. pylori can be isolated under anaerobic conditions. These findings may provide new insight into the physiology of this human pathogen and help to identify the route of H. pylori transmission.
Keywords: bacterial culture, anaerobic conditions, H. pylori isolation
INTRODUCTION
Helicobacter pylori is the leading gastric pathogen that colonizes the human stomach and causes a spectrum of diseases such as chronic active gastritis, gastric ulceration, MALT lymphoma and gastric cancer among others [1–5]. The role of the bacteria as a human pathogen has been confirmed in many parts of the world [6, 7].
Helicobacter pylori is recognized as an inhabitant of the human gastric mucosa. The organism has been regarded as microaerophilic, as its optimal growth occurs in the presence of 5–15% oxygen [8]. In general, primary cultures of H. pylori have less oxygen tolerance with a growth maximum at 3–7% O2 [3, 9]. Most studies with standardized atmospheres for culturing H. pylori have used 2–5% O2, 5–10% (optimal closer to 10%) CO2 and 0–10% H2 [10, 11]. Subcultures of H. pylori can rapidly be adapted to grow aerobically in a standard CO2 mixture (18% O2, 10% CO2) in an incubator [12]. In addition, H. pylori appear to survive in other gas environments such as 10% CO2 alone or variations in oxygen levels [8, 13].
There has been no consensus about the specific oxygen and carbon dioxide needs of Helicobacter pylori. Some investigators do not consider the bacteria a true micro-aerobic, as H. pylori is also a capnophile that grows equally well in vitro under micro-aerobic or aerobic conditions at high bacterial concentrations, and behaves like an oxygen-sensitive microaerophilic at low cell densities [14]. In 1999, Yamaguchi reported that H. pylori strain IK1029 growing under microaerophilic condition was able to survive long incubation periods under anaerobic conditions [15]. Furthermore, H. pylori has also been cultured from the feces [16, 17] that form in the anaerobic large intestine. These reports suggest that H. pylori may grow under anaerobic conditions.
To determine whether H. pylori can grow in both an anaerobic and micro-aerobic environment, we incubated biopsy homogenates from H. pylori infected and not infected patients under each condition. We report here that H. pylori can be isolated as a primary culture under anaerobic conditions as well as under micro-aerobic conditions. We also report that the genotypes of the isolated H. pylori, characterized by traditional PCR, are consistent with the genotypes previously described in this region of the world, independent of culture conditions.
METHODS
1.1. Biopsy specimens
Patients enrolled in Buu Dien Hospital were interviewed and signed informed consent forms before undergoing endoscopic examination to monitor stomach lesions. Patients’ consent forms were approved by a local committee at the Buu Dien Hospital. Gastric biopsies from 27 patients were taken from antrum (AB) and corpus (CB) and flash-frozen immediately in liquid nitrogen for further study without cryopreservative. In addition, gastric juice was collected from all patients and pH was measured using pH paper.
1.2. Microbiological methods
1.2.1, Medium and processing of gastric biopsy samples
Non-selective medium commercial 5% Sheep Blood Agar was used for culturing H. pylori. Gastric biopsies were processed as previously described [18]. The delay between the removal of the specimens from the freezer and the inoculation onto culture media did not exceed 2–4 h. Gastric biopsies were homogenized in 300 µl of sterile 1× PBS and then 30 µl of the suspension was spread on the surface of non-selective blood media. The rest of the biopsy homogenates were used for total genomic DNA purification.
1.2,2, Culture condition
Plates were incubated at 37°C under two different atmospheric conditions: anaerobic and microaerophilic, using the BD Gas Pak™ EZ gas generating container systems (BD Company, Sparks MD) (anaerobic) or the BD Campy container system (microaerophilic), respectively. H. pylori growth was monitored from days 3–7. Bacterial morphology was examined by Gram staining at a magnification of ×1000. Bacterial isolates recovered from anaerobic and microaerophilic conditions were confirmed as H. pylori on the basis of positive urease reaction, typical colony morphology (small, round colonies) and traditional PCR analysis for several bacterial genes including cagA, vacA, hspA, 23SrRNA and the intergenic region between jhp0153 and jhp0152. Furthermore, all H. pylori isolated recovered from anaerobic and microaerophilic conditions were plated again under the same atmospheric conditions to confirm their ability to grow in anaerobic and microaerophilic conditions.
1.3. Molecular methods
1.3.1, DNA purification
Total genomic DNA was purified from the biopsy homogenates using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia CA). Bacterial DNA was purified from strains isolated under anaerobic and microaerobic conditions using the MBI GeneJet Genomic DNA purification Kit (Fermentas, Vilnius, Lithuania). Concentration of extracted purified DNA was determined using the NanoDrop 1000 (Thermo Scientific, Hanover Park IL).
1.3.2, PCR amplification
PCR amplification occurred with 1–2 µl (100–200ng) of purified DNA from biopsies or bacteria. Each PCR reaction contained 10mM Tris-HCl (pH 8.3), 50mM KCl, 1.5mM MgCl2, 200 µM each dNTP, 25pmol each primer, and 2.5 Unit of Taq polymerase. Temperature and step durations are included in Table 1. The reactions were conducted for 30–35 cycles using the Hybaid Thermocycler MBS 0.5G (Thermo Electron Corporation, Marietta OH). The PCR products were confirmed for size and purity on 1.5%–2% agarose gel run with 1× TAE buffer. The primers used in the analysis were shown in the Table 1.
Table 1.
Primers used for PCR and qPCR
| Gene target | Primers | Reference | Temperature and step |
|---|---|---|---|
| cagA | 5’-GTTAARAATRGTGTRAAYGG -3’ 5’-TTTAGCTTCTGATACCGC-3’ |
19 | 94°C 30s, 56°C 30s, 72°C 1min. |
| Empty-site | 5’-CTCTTTTTGTGCCTTTGATTGAA-3’ 5’-CCAAATACATTTTGGCTAAATAAAC-3’ |
21 | 94°C 30s, 54°C 45s, 72°C 1min. |
| hspA | 5’GCTATCTGAAAATTTGATTTCTTTTGC-3’ 5’-TGCGCTATAGTTGTGTCGC-3’ |
23 | 94°C 30s, 52°C 30s, 72°C 2min. |
| Intergenic region |
5'-GTGGCGCGTTTCTTGCAATACC-3' 5'-AACTCGCTCAAAAACTCGGC-3' |
24 | 94°C 30s, 57°C 30s, 72°C 1min. |
| 23S rRNA | 5’-CTCCATAAGAGCCAAAGCCC-3’ 5’-CCACAGCGATGTGGTCTC-3’ |
25 | 94°C 60s, 60°C 45s, 72°C 1min. |
| VacA status | |||
| vacA s-region | 5’-TGAGTTGTTTGATATTGAC-3’ 5’-CAATCTGTCCAATCAAGCGAG-3’ |
22 | 94°C 30s, 50°C 45s, 72°C 45s. |
| vacA m-region | 5’-CTGCTTGAATGCGCCAAAC-3’ 5’-ATGGAAATACAACAAACACAC-3’ |
This study | 94°C 30s, 52°C 30s, 72°C 2min. |
| qPCR | |||
| Hp 16S rRNA | 5’ –AGAGACTAAGCYCTCCAACAAC-3 5’ –AATACTCATTGCGAAGGCGA-3’ |
This study | |
| HP probe 5’ -CATTACTGACGCTGATTGYGC | This study | ||
|
Eubacteria 16s rRNA |
5’ –CAGCAGCCGCGGTRATA-3 5’ –CGYCAATTGBGBAADATTCC-3’ |
26 | |
| Eubacteria probe 5’ –TACGGGAGGCAGCAGT- 3’ |
26 |
cagA
The 3’end of the cagA gene was amplified with two primers, one of them degenerate [19]. The carboxyl terminal portion of CagA protein has multiple phosphorylation sites, and there is a clear difference in the number and type of phosphorylation sites between H. pylori strains isolated from Eastern and Western countries [20]. PCR conditions: 5-min hot start, 25 to 30 cycles as follows: 94°C for 30 s, 56°C for 30 s, 72° C for 1 min. For those strains that were cagA negative, we confirmed the lack of the pathogenicity island by empty-site PCR.
1.3.3, qPCR Methodology
A set of primer, and a TaqMan MGB probe specific for H. pylori were developed based on the H. pylori 16S rRNA gene. Forward primer: 5’-AGAGACTAAGCYCTCCAACAAC; Reverse primer: 5’-AATACTCATTGCGAAGGCGA; and Probe: 5’-TACGGGAGGCAGCAGT. Standards for total bacteria and H. pylori were prepared from the 16S rRNA, which were amplified from H. pylori strain 26695 using the bacterial 8F/1510R primer [21]. PCR products were cloned into the pGEMT easy vector (Promega, Madison, WI), and confirmed by sequencing. The qPCR-mix, with a total volume of 20 µl per well, consisted of 5 µM of forward and reverse primers, 2 µM MGB probe, 10µl Light Cycler 480 Probes Master (Roche), and 1 µl extracted DNA. The thermal cycle program consisted of 45 cycles of amplification at 95°C for 10s, 54°C for 30s, and then 72°C for 20s, with an initial cycle of 95°C for 10 min. The assays were performed using the Roche LightCycler 480 II PCR system, run in duplicate, and results analyzed using the LightCycler480 II program (Roche). We used 10-fold dilutions of the cloned 16S rRNA gene from H. pylori strain 26695 to build standard curves to estimate the quantity of H. pylori. We also estimated the quantity of total bacteria from DNA purified from gastric biopsies using a previously described method [21].
1.3.4, PCR amplification of virulence markers
Empty site
The test for empty-site, related to the presence of the PAI Island and cagA gene in H. pylori genome, was performed in cagA negative strains [22]. Determination of vacA genotypes. The vacA genotype (s1/s2 or m1/m2) of each strain was evaluated according to the method of [23]. The primers used in this study for vacA m1/m2 genotyping were designed in our laboratory for this study. HspA (heat shock protein gene). The fragment of H. pylori hspA gene was amplified as previously reported [24].
Intergenic region between jhp0153 and jhp0152
A PCR reaction amplified an intergenic region from the H. pylori genome as previously reported [25]. Based on the product size amplified, we could identify H. pylori strains of African origin by the distinctive 180bp insert that they carry in this intergenic region [25].
23S rRNA
Full length of 23S rRNA gene was amplified with primers as previously described [26].
1.4. H. pylori infection confirmation
The H. pylori status of the patients was confirmed based on the results of qPCR and culture-based methods. The patient's H. pylori status was considered positive if qPCR results were positive in the purified DNA extracted from the gastric biopsy and the H. pylori/total bacteria ratio assessed by qPCR was >30% based on our validation experiment that included 25 well known positive H. pylori subjects and 20 well known H. pylori negative subjects (data not shown). Patients were considered H. pylori negative, if PCR results performed on purified gastric biopsy DNA were negative and the ratio of H. pylori to total bacteria by qPCR was <30%.
1.5 Non-H. pylori organisms identification
Single colonies of non-Helicobacter pylori appearing on the same blood-agar plates with H. pylori were selected from the agar surface and purified by sub-culturing several times using TSA agar plates with 5% sheep blood, incubated under anaerobic and aerobic conditions, and subsequently incubated at 37°C for 48 hours prior to DNA extraction. We next performed PCR of the 16s rRNA gene using bacteria universal primers previously described [27]. Next we performed purification of the PCR product, and 16S rRNA sequencing using Sanger method (Macrogen, NYC, US) [22, 26]. DNA was extracted using genomic DNA purification kit (Fermentas K0722).
RESULTS AND DISCUSION
2.1 Demographic and clinical characteristics of patients
Over a study period of 5 months, from December 2010 to April 2011, 54 gastric mucosal biopsy specimens were obtained from 27 patients with gastric bleeding (15 male, 12 female). The mean age of the group was 36.3 years, with a range of 25 to 57 years of age. Gastric juice pH was found as expected to be acidic for all patients with a mean value of 2 and a range between 1 and 4, except for one patient with a gastric juice pH of 8.0.
2.2 H. pylori infection of the patients confirmed by qPCR
The H. pylori infection of the patients was determined by qPCR using genomic DNA extracted from the gastric biopsies. It was confirmed by calculating the proportion of H. pylori present in the biopsy to the total number of bacteria estimated by qPCR in the same sample. We defined as a threshold of positivity that the proportion of H. pylori was greater than or equal to 30%. The H. pylori infection status of patients is presented in Table 2. Twenty-one (77.8%) patients were determined as H. pylori positive and 6 patients as H. pylori negative by quantitative PCR. Nearly all subjects with at least one biopsy sample positive by culture were also positive by qPCR with the exception of one patient (6.25%). In addition, of the 23 biopsies with culture positive, 4 (17.4%) were qPCR negative. These discrepancies are due to the patchy distribution of the infection. Regardless of the criteria used to define H. pylori status, biopsies obtained from the antrum were more informative of the H. pylori status than biopsies obtained from the corpus. However, the difference in reliable H. pylori status determined from these body sites was not significant. Our results confirmed the relevance of molecular methodologies for the diagnostic of H. pylori [28].
Table 2.
H. pylori infection of the patients confirmed by qPCR and culture method
| Biopsy location | qPCR positive | Culture positive | ||
|---|---|---|---|---|
| n | % | n | % | |
| Antrum and Corpus | 8 | 29.6 | 7 | 25.9 |
| Antrum only | 10 | 37.0 | 6 | 22.2 |
| Corpus only | 3 | 11.1 | 3 | 11.1 |
| Either | 21 | 77.8 | 16 | 59.3 |
2.3 Primary isolation of H. pylori under anaerobic and microaerophilic conditions
Table 3 shows the recovery rates of H. pylori strains from biopsies obtained from 21 H. pylori infected-patients with gastric bleeding under microaerophilic and anaerobic conditions. The results indicate that an anaerobic environment coupled with blood agar provides growth conditions that yield the highest H. pylori recovery rate when compared with microaerophilic conditions. All the micro-aerobic positive samples were also positive in anaerobic culture conditions. The recovery of H. pylori under anaerobic was higher than under microaerophilic conditions (81.3% vs.31.3%, p value <0.01 Fisher Exact Test). All the isolated colonies under anaerobic conditions were able to grow under the same anaerobic conditions outside of the primary isolation using the same methodology for generating anaerobic conditions.
Table 3.
Recovery rates of H. pylori strains in 16 culture positive patients under microaerophilic and anaerobic conditions
| Biopsy location | Microaerobic environment | Anaerobic environment | ||
|---|---|---|---|---|
| n | % | n | % | |
| Antrum and corpus | 0 | 0 | 7 | 43.8 |
| Antrum only | 3 | 18.8 | 4 | 25.0 |
| Corpus only | 2 | 12.5 | 2 | 12.5 |
| Either | 5 | 31.3* | 13 | 81.3* |
Chi square Fisher exact test p values < 0.01
Non-H. pylori bacteria strains appeared to occur in the same plates with H. pylori under anaerobic conditions. They were recovered and identified by 16S rRNA analysis as facultative anaerobic bacteria Streptococcus spp. (S. australis, S. infantis and Bacillus spp. (B. licheniformis, B. subtilis, B. cereus, B. megaterium). The remaining non-H. pylori bacterial isolates were identified as Ochrobactrum intermedium, Rothia mucilaginosa and R. dentocariosa.
2.4 Genotyping H. pylori isolates
Of the 23 isolated H. pylori strains, DNA was purified from 20 (87%) H. pylori isolates obtained from 14 of the 16 patients with a positive H. pylori status. Each strain was genotyped (Table 4). In six out of the 14 patients (42.9%), H. pylori strains were recovered from both sites of the stomach (antrum and corpus). For the remaining 8 patients, H. pylori strains were recovered from only one site. The presence of CagA positive strains is nearly universal among patients from East Asian countries colonized with H. pylori, according to Yamaoka et al and Truong et al [29, 30], and as expected, 80.0% (16 of 20) of H. pylori isolates from this Vietnamese population were cagA positive. We sequenced the 3’ variable region and confirmed that all the strains possess the Eastern D motif at the carboxyl terminal region of CagA, as has been previously reported from East Asian countries [20, 31]. Interestingly, isolates obtained from two patients (8 and 21) lacked the cagA gene according to PCR analysis. However, these same isolates showed evidence of carrying the Cag pathogenicity island. We did not test further to determine if those strains harbored a partial Type Four Secretion System (T4SS).
Table 4.
Genetic characterization of H.pylori strains isolated from gastritis patients
| Patien t |
Biopsy location |
Intergenic region* |
cagA | Empty site** | hspA | vacA s/m | Culture conditions |
|---|---|---|---|---|---|---|---|
| 1 | A/C | − | + | + | s1/m2 | Anaerobic | |
| 3 | A/C | − | + | + | s1/m1/m2 | Micro/Ana | |
| 4 | A | − | + | + | s1/m1 | Anaerobic | |
| 5 | A/C | − | + | + | m1/m2 | Anaerobic | |
| 7 | A/C | − | + | + | s1/m2 | Anaerobic | |
| 8 | C | − | − | − | + | s1/m2 | Micro/Ana |
| 17 | A | − | − | + | + | s1/m2 | Anaerobic |
| 19 | C | − | + | + | s1/m1/m2 | Micro/Ana | |
| 21 | A | − | − | − | + | s1/m2 | Anaerobic |
| 23 | A | − | + | + | s1/m2 | Micro/Ana | |
| 24 | A/C | − | + | + | s1/m2 | Micro/Ana | |
| 25 | A | − | − | + | + | s1/m2 | Anaerobic |
| 26 | A/C | − | + | + | s1/m1 | Anaerobic | |
| 27 | A | − | + | + | s1/m1 | Anaerobic |
A= antrum
C= corpus
s= signal sequence
m=middle region
Micro=micro-aerobic conditions
Ana=anaerobic conditions
Negative for the 180bp insert
Empty-site PCR was performed only on cagA negative strains in order to determine presence or absence of the cag pathogenicity island. The presence of a band in the empty site PCR represents the lack of Pathogenicity Island.
All 20 H. pylori strains included the16 strains that tested positive for the cagA gene were vacA positive. Of these strains, twelve (75%) showed the s1/m2 genotype and 4 showed the s1/m1 genotype. Some of the H. pylori strains showed multiple alleles, despite single colonies being picked. DNA was purified from only one of these strains for testing by PCR, thus, we cannot rule out the possibility of mixed infection in those patients with multiples vacA alleles. A possible mixed infection may also explain the case of patient 17 with cagA positivity but empty-site positivity. All H. pylori isolates in the study were confirmed to bear the intergenic region between jhp0153 and jhp0152 in their genome. However, none of the H. pylori isolates carried the 180bp insert that has been associated with strains of African origin [24]. The intergenic region we observed has been reported mostly in H. pylori strains isolated from East Asian countries such as Japan and Korea [24].
Humans are the major reservoir of H. pylori, where the bacteria appears to occur along certain areas of the gastrointestinal tube, including dental plaque, the esophagus, stomach, and gastric metaplasia into small intestine and large intestine [7, 17, 32–35].
In this study we provide evidence that H. pylori not only survives under anaerobic conditions, as Yamaguchi described [15], but that it can grow on non-selective blood agar under the anaerobic conditions generated by the BD Gas Pak. Furthermore, the genotypes of the H. pylori recovered are consistent with the genotypes previously described in Vietnam [36–38].
H. pylori acquisition occurs in early infancy via oral-oral or oral-fecal routes [39–41]. In addition, H. pylori have been isolated by culture method from human feces under micro-aerobic conditions, and now this study has confirmed that H. pylori can also grow in anaerobic conditions. In vivo, the bacteria might colonize the anaerobic large intestine microbiota or be washed from the upper digestive tract (stomach) and distributed along the human digestive tube, where H. pylori continues to grow approximately 3–4 hours within the large anaerobic intestine [42, 43] before being excreted in feces into the external environment. The fecal-oral transmission via water - or waterborne transmission - might be probable in the spread of infection in human communities, especially in crowded ones with inadequate sanitation.
The findings of this study may provide new insight into the physiology of this fastidious human pathogen and inform future epidemiological studies covering the route of Helicobacter pylori's transmission.
Highlights.
We were able to growth Helicobacter pylori under anaerobic conditions.
The use of anaerobic pack yield better recovery of H. pylori than Campy pack.
No difference in genotype or phenotype was observed between the bacteria growth in either condition.
Acknowledgments
This research was supported in part by RO1GM63270 by the National Institutes of Health and in part by Research project no. 50/2010/HD-NDT from the Ministry of Sciences and Technology, Vietnam. We thank Thomas Battaglia for his revision of the English style of this manuscript.
Footnotes
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
All the authors declared that they do not have any conflict of interest.
References
- 1.Kidd M, Modlin IM. A century of Helicobacter pylori: Paradigms lost-paradigms regained. Digestion. 1998;59:1–15. doi: 10.1159/000007461. [DOI] [PubMed] [Google Scholar]
- 2.Warren IR. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet. 1983;1:1273–1275. [PubMed] [Google Scholar]
- 3.Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1984;1:1311–1315. doi: 10.1016/s0140-6736(84)91816-6. [DOI] [PubMed] [Google Scholar]
- 4.Nomura A, Stemmermann GN, Chyou PH, Kato I, Perez-Perez GI, Blaser MJ. Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii. New Engl J Med. 1991;325:1132–1136. doi: 10.1056/NEJM199110173251604. [DOI] [PubMed] [Google Scholar]
- 5.Biological agents. Volume 100 B. A review of human carcinogens. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. IARC Monogram Eval Carcinog Risks Hum. 2012;100(Pt B):1–441. [PMC free article] [PubMed] [Google Scholar]
- 6.Go MF. Review article: Natural history and epidemiology of Helicobacter pylori infection. Aliment Pharmacol Ther. 2002;16:3–15. doi: 10.1046/j.1365-2036.2002.0160s1003.x. [DOI] [PubMed] [Google Scholar]
- 7.Brown LM. Helicobacter pylori epidemiology and routes of transmission. Epidemiol Rev. 2000;22:283–297. doi: 10.1093/oxfordjournals.epirev.a018040. [DOI] [PubMed] [Google Scholar]
- 8.Goodwin CS, Armstrong JA. Microbiological aspects of Helicobacter pylori (Campylobacter pylori) Eur J Clin Microbiol. 1990;9:1–13. doi: 10.1007/BF01969526. [DOI] [PubMed] [Google Scholar]
- 9.Langenberg M, Tytgat GNJ, Schipper MEI, Rietra PJGM, Zanen HC. Campylobacterlike organisms in the stomach of patients and individuals. Lancet. 1984;323:1348–1349. [Google Scholar]
- 10.Andersen LP, Wadström T. Helicobacter pylori: Physiology and Genetic. In: Harry LT Mobley, George L Mendz, Stuart L Hazell., editors. Editor Information. Washington (DC): ASM Press; 2001. ISBN-10: 1-55581-213-9. [Google Scholar]
- 11.Kusters JG, van Vliet AHM, Kuipers EJ. Pathogenesis of Helicobacter pylori Infection. Clinical Microbiol Rev. 2006;19:449–490. doi: 10.1128/CMR.00054-05. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Tompkins DS, Dave J, Mapstone NP. Adaptation of Helicobacter pylori to aerobic growth. Eur. J. Clin. Microbiol. Infect. Dis. 1994;13:409–412. doi: 10.1007/BF01971999. [DOI] [PubMed] [Google Scholar]
- 13.West AP, Millar MR, Tompkins DS. Effect of physical environment on survival of Helicobacter pylori. J Clin Pathol. 1992;45:228–231. doi: 10.1136/jcp.45.3.228. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Bury-Moné S, Kaakoush NO, Asencio C, Mégraud F, Thibonnier M, De Reuse H, Mendz GL. Is Helicobacter pylori a True Microaerophile? Helicobacter. 2006;11:296–303. doi: 10.1111/j.1523-5378.2006.00413.x. [DOI] [PubMed] [Google Scholar]
- 15.Yamaguchi H, Osaki T, Takahashi M, Taguchi H, Kamiya S. Colony formation by Helicobacter pylori after long-term incubation under anaerobic conditions. FEMS Microbiol Lett. 1999;175:107–111. doi: 10.1111/j.1574-6968.1999.tb13608.x. [DOI] [PubMed] [Google Scholar]
- 16.Thomas JE, Gibson GR, Darboe MK, Dale A, Weaver LT. Isolation of Helicobacter pylori from faeces. Lancet. 1992;340:1194–1195. doi: 10.1016/0140-6736(92)92894-l. [DOI] [PubMed] [Google Scholar]
- 17.Kelly SM, Pitcher MC, Farmery SM, Gibson GR. Isolation of Helicobacter pylori from feces of patients with dyspepsia in the United Kingdom. Gastroenterology. 1994;107:1671–1674. doi: 10.1016/0016-5085(94)90806-0. [DOI] [PubMed] [Google Scholar]
- 18.Peek RM, Jr, Miller GG, Tham KT, Pérez-Pérez GI, Cover TL, Atherton JC, Dunn GD, Blaser MJ. Detection of Helicobacter pylori gene expression in human gastric mucosa. J Clin Microbiol. 1995;33:28–32. doi: 10.1128/jcm.33.1.28-32.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Panayotopoulou EG, Sgouras DN, Papadakos K, Kalliaropoulos A, Papatheodoridis G, Mentis AF, et al. Strategy to characterize the number and type of repeating EPIYA phosphorylation motifs in the carboxyl terminus of CagA protein in Helicobacter pylori clinical isolates. J Clin Microbiol. 2007;45:488–495. doi: 10.1128/JCM.01616-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Hatekeyama M, Higashi H. Helicobacter pylori CagA: a new paradigm for bacteria carcinogenesis. Cancer Sci. 2005;96:835–843. doi: 10.1111/j.1349-7006.2005.00130.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Gao Z, Perez Perez GI, Chen Y, Blaser MJ. Quantification of major human cutaneous bacterial and fungal populations. J Clin Microbiol. 2010;48:3575–3581. doi: 10.1128/JCM.00597-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Chung C, Olivares A, Torres E, Yilmaz O, Cohen H, Perez-Perez GI. Diversity of VacA intermediate region among Helicobacter pylori strains from several regions of the world. J. Clin. Microbiol. 2010;48:690–696. doi: 10.1128/JCM.01815-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Atherton JC, Peek RM, Tham KT, Cover TL, Blaser MJ. Clinical and pathological importance of heterogeneity in vacA, the vacuolating cytotoxin gene of Helicobacter pylori. Gastroenterol. 1997;112:92–99. doi: 10.1016/s0016-5085(97)70223-3. [DOI] [PubMed] [Google Scholar]
- 24.Kansau I, Guillain F, Thiberge JM, Labigne A. Nickel binding and immunological properties of the C-terminal domain of the Helicobacter pylori GroES homologue (HspA) Mol Microbiol. 1996;22:1013–1023. doi: 10.1046/j.1365-2958.1996.01536.x. [DOI] [PubMed] [Google Scholar]
- 25.McNulty SL, Mole BM, Dailidiene D, Segal I, Ally R, Mistry R, Secka O, Adegbola RA, Thomas JE, Lenarcic EM, Peek RM, Berg DE, Forsyth MH. Novel 180- and 480-base-pair Insertions in African and African-American strains of Helicobacter pylori. J. Clin. Microbiol. 2004;42:5658–5663. doi: 10.1128/JCM.42.12.5658-5663.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Agudo S, Pérez-Pérez GI, Alarcón T, López Brea M. High prevalence of clarithromycin-resistant Helicobacter pylori strains and risk factors associated with resistance in Madrid, Spain. J Clin Microbiol. 2010;48:3703–3707. doi: 10.1128/JCM.00144-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Gao Z, Tseng CH, Pei Z, Blaser MJ. Molecular analysis of human forearm superficial skin bacterial biota. Proc Natl Acad Sci U S A. 2007;104:2927–2932. doi: 10.1073/pnas.0607077104. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Mégraud F. The most important diagnostic modalities for Helicobacter pylori, now and in the future. Eur J Gastroenterol Hepatol. 2012;9(Suppl 1):S13–S15. [PubMed] [Google Scholar]
- 29.Yamaoka Y, Kato M, Asaka M. Geographic differences in gastric cancer incidence can be explained by differences between Helicobacter pylori strains. Intern Med. 2008;47:1077–1083. doi: 10.2169/internalmedicine.47.0975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Truong XB, Mai VTC, Tanaka H, Ly LT, Thong TM, Hai HH, Long DV, Furumatsu K, Yoshida M, Kutsumi H, Azuma T. Diverse characteristics of the cagA gene of Helicobacter pylori strains collected from patients from Southern Vietnam with gastric cancer and peptic ulcer. J Clin Microbiol. 2009;47:4021–4028. doi: 10.1128/JCM.00504-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Azuma T. Helicobacter pylori CagA protein variation associated with gastric cancer in Asia. J. Gastroenterol. 2004;39:97–103. doi: 10.1007/s00535-003-1279-4. [DOI] [PubMed] [Google Scholar]
- 32.Rasmussen LT, de Labio RW, Neto AC, Silva LC, Queiroz VF, Smith MAC, Payão SLM. Detection of Helicobacter pylori in gastric biopsies, saliva and dental plaques of dyspeptic patients from Marília, São Paulo, Brazil: presence of vacA and cagA gene. J. Venom. Anim. Toxins incl. Trop. Dis. 2012;18:180–187. [Google Scholar]
- 33.Momtaz H, Souod N, Dabiri H, Sarshar M. Study of Helicobacter pylori genotype status in saliva, dental plaques, stool and gastric biopsy samples. World J Gastroenterol. 2012;18:2105–2111. doi: 10.3748/wjg.v18.i17.2105. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.Agarwal S, Jithendra KD. Presence of Helicobacter pylori in subgingival plaque of periodontitis patients with and without dyspepsia, detected by polymerase chain reaction and culture. J Indian Soc Periodontol. 2012;16:398–403. doi: 10.4103/0972-124X.100919. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Bakri MM. Invasive test for Helicobacter pylori infection in symptomatic patients and healthy volunteers. Pak J Physiol. 2011;8:10–12. [Google Scholar]
- 36.Pounder RE, Ng D. The prevalence of Helicobacter pylori infection in different countries. Alimen Pharmacol Ther. 1995;9:33–39. [PubMed] [Google Scholar]
- 37.Nguyen LT, Uchida T, Murakami K, Fujioka T, Moriyama M. Helicobacter pylori virulence and the diversity of gastric cancer in Asia. J Med Microbiol. 2008;57:1445–1453. doi: 10.1099/jmm.0.2008/003160-0. [DOI] [PubMed] [Google Scholar]
- 38.Nguyen LT, Uchida T, Tsukamoto Y, Dung TT, Long T, Bang HM, Song HL, Ky DT, Dung DH, Hai HH, Matsuhisa T, Okimoto T, Kodama M, Murakami K, Fujioka T, Yamaoka Y, Moriyama M. Helicobacter pylori infection and gastroduodenal diseases in Vietnam: a cross-sectional, hospital-based study. BMC Gastroenterology. 2010;10:114–120. doi: 10.1186/1471-230X-10-114. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Mégraud F. Transmission of Helicobacter pylori: faecal–oral versus oral–oral route. Aliment. Pharmacol Ther. 1995;9:85–91. [PubMed] [Google Scholar]
- 40.Cave DR. Transmission and epidemiology of Helicobacter pylori. Am. J. Med. 1996;100:2S–17S. doi: 10.1016/s0002-9343(96)80224-5. (1996) [DOI] [PubMed] [Google Scholar]
- 41.van Duynhoven YT, de Jonge R. Transmission of Helicobacter pylori: a role for food? Bull World Health Organ. 2001;79:455–460. [PMC free article] [PubMed] [Google Scholar]
- 42.Kim SK. Small intestine transit time in the normal small bowel study. American Journal of Roentgenology Ther Nucl Med. 1968;104:522–524. doi: 10.2214/ajr.104.3.522. [DOI] [PubMed] [Google Scholar]
- 43.Ghoshal UC, Sengar V, Srivastava D. Colonic Transit Study Technique and Interpretation: Can These Be Uniform Globally in Different Populations with Non-uniform Colon Transit Time? J Neurogastroenterol Motil. 2012;18:227–228. doi: 10.5056/jnm.2012.18.2.227. [DOI] [PMC free article] [PubMed] [Google Scholar]