Simultaneous Amplification of Two Bacterial Genes: More Reliable Method of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Detection in Microbial Rich Dental Plaque Samples

Polymerase Chain reaction (PCR) assay is considered superior to other methods for detection of Helicobacter pylori (H. pylori) in oral cavity; however, it also has limitations when sample under study is microbial rich dental plaque. The type of gene targeted and number of primers used for bacterial detection in dental plaque samples can have a significant effect on the results obtained as there are a number of closely related bacterial species residing in plaque biofilm. Also due to high recombination rate of H. pylori some of the genes might be down regulated or absent. The present study was conducted to determine the frequency of H. pylori colonization of dental plaque by simultaneously amplifying two genes of the bacterium. One hundred dental plaque specimens were collected from dyspeptic patients before their upper gastrointestinal endoscopy and presence of H. pylori was determined through PCR assay using primers targeting two different genes of the bacterium. Eighty-nine of the 100 samples were included in final analysis. With simultaneous amplification of two bacterial genes 51.6% of the dental plaque samples were positive for H. pylori while this prevalence increased to 73% when only one gene amplification was used for bacterial identification. Detection of H. pylori in dental plaque samples is more reliable when two genes of the bacterium are simultaneously amplified as compared to one gene amplification only.     

Food-Grade Expression of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> UreB Subunit in <Emphasis Type="Italic">Lactococcus lactis</Emphasis> and its Immunoreactivity

Helicobacter pylori is the principal cause of chronic active gastritis, peptic ulcer, and gastric cancer. To develop an oral vaccine against H. pylori infection, we had expressed the H. pylori ureB gene (Genbank accession no. FJ436980) in nisin-controlled expression vectors using Lactococcus lactis NZ3900 as host. The ureB gene was amplified by PCR from a H.pylori strain MEL-Hp27. Then the ureB gene was fused translationally downstream of the nisin-inducible promoter nisA in a L. lactis plasmid pNZ8149. Lactose utilization based on the complementation of the lacF gene was used as a dominant selection marker for the food-grade expression system employing L. lactis NZ3900. The conditions of UreB expression in this system were optimized by orthogonal experiment. The optimized conditions have been determined as follows: induction of expression was carried out at the cells density of OD₆₀₀ ≈ 0.4 with 25 ng/ml nisin, and harvest after 5 h. The maximum percentage of recombinant UreB was estimated to be 7% of total soluble cellular proteins and the yield was 12.9 μg/ml. Western blot demonstrated that the UreB protein was expressed in the L. lactis transformant and had favorable immunoreactivity. These results indicated that the lactococci-derived vaccines could be promising candidates as alternative vaccine strategies for preventing H. pylori infection.     

Human lactoferrin increases <Emphasis Type="Italic">Helicobacter pylori</Emphasis> internalisation into AGS cells

Helicobacter pylori has high global infection rates and can cause other undesirable clinical manifestations such as duodenal ulcer (DU) and gastric cancer (GC). Frequencies of re-infection after therapeutic clearance and rates of DU versus GC vary geographically and differ markedly between developed and developing countries, which suggests additional factors may be involved. The possibility that, in vivo, lactoferrin (Lf) may play a subtle role in modulating micronutrient availability or bacterial internalisation with implications for disease etiology is considered. Lf is an iron binding protein produced in mammals that has antimicrobial and immunomodulatory properties. Some bacteria that regularly colonise mammalian hosts have adapted to living in high Lf environments and we investigated if this included the gastric pathogen H. pylori. We found that H. pylori was able to use iron from fully iron-saturated human Lf (hLf) whereas partially iron-saturated hLf (apo) did not increase H. pylori growth. Instead, apo-hLf increased adherence to and internalisation of bacteria into cultured epithelial cells. By increasing internalisation, we speculate that apo-human lactoferrin may contribute to H. pylori’s ability to persistence in the human stomach, an observation that potentially has implications for the risk of H. pylori-associated disease.     

Transgenic Peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) Expressing the Urease Subunit B Gene of <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

Helicobacter pylori (H. pylori) has been identified as the main pathogenic factors of chronic gastritis and peptic ulcer, and the Class I carcinogen of gastric cancer by WHO. Vaccine has become the most effective measure to prevent and cure H. pylori infection. The UreB is the most effective and common immunogen of all strains of H. pylori and may stimulate the immunoresponse protecting the human body against the challenge of H. pylori. UreB antigen gene was cloned into the binary vector pBI121 which contains a seed-specific promoter Oleosin of peanut and a kanamycin resistance gene, and then UreB gene was transformed into peanut embryo leaflets by Agrobacter-mediated method. The putative transgenic plants were examined for the presence of UreB in the nuclear genome of peanut plants by PCR analysis. Expression of UreB gene in plants was identified by RT-PCR and Western blot analysis. These results suggest that the UreB transgenic peanut can be potentially used as an edible vaccine for controlling H. pylori.     

Association of <Emphasis Type="Italic">iceA</Emphasis> and <Emphasis Type="Italic">babA</Emphasis> genotypes in <Emphasis Type="Italic">Helicobacter pylori</Emphasis> strains with patient and strain characteristics

Data on the geographic prevalence of Helicobacter pylori iceA and babA alleles in Eastern Europe are still relatively scant. The aim of this study was to evaluate the prevalence of iceA and babA genotypes in Bulgarian symptomatic patients. The iceA and babA genotypes were evaluated by PCR with pure cultures in strains from 196 and 181 patients, respectively. Mixed infections were found in 10.2% of all 196 patients. Prevalence of H. pylori genotypes in patients with single-strain infections was 69.3% for iceA1, 30.7% for iceA2, 82.4% for cagA ⁺, 89.2% for vacA s1, 10.8% for vacA s2, 39.8% for vacA m1, 60.2% for vacA m2 and 48.8% for babA2. Within the iceA1 positive strains, 94.3% and 88.5% were also vacA s1a and cagA positive, respectively. Of the babA2 positive strains, 100.0%, 92.4% and 72.2% were also vacA s1a, cagA and iceA1 positive, respectively. Ulcer patients had more often strains with cagA positive status and vacA s1a allele. Although neither iceA1 nor babA2 were more common in ulcer patients, the combination of both alleles was more frequent (48.1%) in the ulcer patients than in the rest (28.7%). Clarithromycin susceptible strains had more often iceA1 allele (74.4%) than the resistant strains (55.3%). In conclusion, the results demonstrated a high prevalence of virulent H. pylori in Bulgaria. Both iceA1 and babA2 genotypes were associated with other virulence factors of H. pylori and, in addition, the iceA1 allele was associated with the strain susceptibility.     

Non-Invasive Serological Detection of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Antigens in Adult Hong Kong Chinese Non-Ulcer Dyspepsia Patients

Objective The aim of our study was to evaluate the accuracy of the serology test kit (Signify H. pylori test), an office-based serological test and to compare relative performances with endoscopy, including biopsy urease tests (Clotest), histology, and culture when applied to dyspeptic adult patients in Hong Kong Chinese. Methods Prospective clinical trial was conducted at the Alice Ho Miu ling Nethersole Hospital (AHNH) outpatient endoscopy center from June to December 2004 and included adult Chinese patients who attended the endoscopy center. A research nurse interviewed consenting patients and whole blood samples were obtained via simple fingerstick were then and transferred to Signify test kits. The results were read after 5 minutes. The presence of two distinct lines within the result-reading window was treated as a positive result; a single distinct line was treated as a negative result. All patients underwent upper gastroduodenal endoscopy, and antral/antrum gastric biopsy specimens were taken and then processed for microbiological cultures. Results A total of 384 patients were recruited in the study and 131 (34.7%) patients were tested positive for H. pylori. Among them, 62 patients (47.3%) were detected by the Signify H. pylori test. The Signify serology test kit was 52.7% sensitive, 87.9% specific, and 75.7% accurate, respectively in the current study. Conclusion The Signify H. pylori test showed a sub-optimal correlation between the presence of H. pylori infection and the serological test results in a Hong Kong Chinese population.     

<Emphasis Type="Italic">Braf</Emphasis>, <Emphasis Type="Italic">Kras</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis> epigenetic changes-associated chronic gastritis in Egyptian patients with and without gastric cancer

We aimed to study MLH1 and MGMT methylation status in Helicobacter pylori-associated chronic gastritis in Egyptian patients with and without gastric cancer. 39 patients were included in our study. They were divided into 2 groups; patients without (group I) and with gastric adenocarcinoma (group II). Patients were subjected to clinical examination, abdominal ultrasound and upper endoscopy for gastric biopsy. Biopsies were subjected to urease test, histological examination, and DNA purification. H. pylori, Braf, Kras, MLH1 and MGMT methylation were assessed by quantitative PCR. DNA sequencing was performed to assess Braf and Kras genes mutation. qPCR of H. pylori was significantly higher in patients with adenocarcinoma (group II) than those without adenocarcinoma (group I); with a p < 0.001 as well as in patients with age above 50 years with a p value = 0.008. By applying logistic regression analysis it was reported that the H. pylori qPCR is a significant predictor to the adenocarcinoma with OR = 1.025 (95 % CI: 1. 002–1.048), with sensitivity of 90 % and specificity of 100 %. Adenocarcinoma patients had a significantly higher mean age and levels of H. Pylori, Braf, K-ras, methylated MGMT and methylated MLH1 than those of gastritis patients. DNA sequence analysis of Braf (codon 12) and Kras (codon 600) had genes mutation in gastric adenocarcinoma versus chronic gastritis. Conclusion: H. pylori may cause epigenetic changes predisposing the patients to cancer stomach. Estimation of H. pylori by qPCR can be a good predictor to adenocarcinoma. Braf and Kras genes mutation were reveled in gastritis and adenocarcinoma patients.     

The <Emphasis Type="Italic">GPX1</Emphasis> Pro<Superscript>198</Superscript>Leu polymorphism in gastric cancer patients with and without <Emphasis Type="Italic">Helicobacter pylori</Emphasis> infection

Helicobacter pylori infection could induce oxidative stress. Oxidative stress is involved in the pathogenesis of gastric diseases. Glutathione peroxidase 1 (GPX1), is part of the enzymatic antioxidant defense, preventing oxidative damage. The aim of the present study was to assess the association of GPX1 Pro¹⁹⁸Leu genotypes with gastric cancer in patients with and without H. pylori infection in a population of Northern Iran. The present case-control study consisted of 50 patients with gastric cancer and 78 cancer-free subjects as controls. Extraction of DNA was performed on bioptic samples and the GPX1 genotypes were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The frequencies of GPX1 Pro/Pro, Pro/Leu and Leu/Leu genotypes in controls were 21.8, 71.8 and 6.4%, respectively. However, in gastric cancer patients, the frequencies of 34, 56 and 10% were observed for Pro/Pro, Pro/Leu and Leu/Leu genotypes, respectively (p?=?0.185). In 38 (76%) patients infected with H. pylori, the frequencies of Pro and Leu alleles were 94.7 and 3.3%, respectively. There was a higher frequency of combined genotype of Pro/Leu?+?Leu/Leu (94.7%) in H. pylori positive patients than that in patients without H. pylori infection (75%, p?=?0.047). The presence of this genotype tended to increase the risk of H. pylori related gastric cancer by 5.88–fold (p?=?0.069) in our population. Our findings indicated the absence of association between the GPX1 Pro¹⁹⁸Leu polymorphism and the risk of gastric cancer in an Iranian population. However, we detected an association between H. pylori related gastric cancer with GPX1 Pro/Leu?+?Leu/Leu genotype.     

<Emphasis Type="Italic">Helicobacter pylori</Emphasis> outer membrane vesicles involvement in the infection development and <Emphasis Type="Italic">Helicobacter pylori</Emphasis>-related diseases

Helicobacter pylori - (H. pylori) play a role in the pathogenesis of gastritis, gastric and duodenal ulcers as well as gastric cancer. A possible involvement of outer membrane vesicles (OMVs) produced by H. pylori in the distribution of bacterial antigens through the gastric epithelial barrier and their role in the development of local and systemic host inflammatory and immune responses has been suggested. OMVs contain various biologically active compounds, which internalize into host cells affecting signaling pathways and promoting apoptosis of gastric epithelial and immunocompetent cells. OMVs-associated H. pylori virulence factors may strengthen or downregulate the immune responses leading to disease development. This review describes the biological importance of H. pylori OMVs and their role in the course of H. pylori infections, as well as H. pylori related local and systemic effects.     

Core Genome Haplotype Diversity and <Emphasis Type="Italic">vacA</Emphasis> Allelic Heterogeneity of Chinese <Emphasis Type="Italic">Helicobacter pylori</Emphasis> Strains

The human gastric pathogen, Helicobacter pylori, has co-evolved with its host and established itself in the human stomach possibly millions of years ago. Therefore, the diversity of this bacterium is important in its clinical manifestations. Our aim has been to evaluate the genetic diversity of 40 H. pylori clinical isolates from four different parts of China. The methods of multi-locus sequence typing and vacA allele genotyping were used to assess their genetic diversity. To discriminate MLST, the vacA genotype method was used to identify strains. Patients from the northern, eastern, southern, and southwestern parts of China were recruited randomly from the cities of Beijing, Shanghai, Guangzhou, and Chongqing, respectively. Most of the sequence types are new and have never been reported in the database of the H. pylori multi-locus sequence typing system. The most prevalent vacA genotype in patients was s1a/m2 (80.0%), followed by s1b/m2 (17.5%). In contrast, the s1a/m1 genotype was scarcely represented (2.5%). The vacA genotype varied for each ST. These results showed that the MLST method offers high resolution of the H. pylori isolates in China when compared to vacA genotyping. The vacA allelic s1a has been correlated with the peptic ulcer. Because of the paucity of data on human isolates due to the absence of systematic investigations of H. pylori in China, the data provide useful information for understanding the epidemiology of H. pylori in China from the viewpoint of nucleotide sequence databases.     

<Emphasis Type="Italic">PARP-1 Val762Ala</Emphasis> polymorphism,CagA<Superscript>+</Superscript><Emphasis Type="Italic">H. pylori</Emphasis> infection and risk for gastric cancer in Han Chinese population

Introduction PARP-1 plays important role in the BER (base excision repair) and maintenance of genomic integrity. Previous study found the Val762Ala genetic variant in the PARP-1 gene contributed to susceptibility of some cancers and decreased PARP-1 enzyme activity in response to oxidative damage. Helicobacter pylori (H. pylori) infection was thought to be one of the major causes of gastric cancer. In this study, we investigated the association between the PARP-1 Val762Ala polymorphism, CagA⁺ H. pylori infection, and the risk for gastric cancer. Methods This hospital-based, case–control study was performed involving 556 individuals (236 cases with gastric cancer and 320 controls without evidence of neoplasm and gastrointestinal disease) using a PCR-RFLP method. Chi-square test and logistic regression analysis were used to count OR and 95% CI. Results 762Ala/Ala genotype was overrepresented in the cases (16.9%) compared with controls (10.3%), (OR, 1.942; 95% CI, 1.157–3.257, P = 0.011). Multivariate analysis showed that two factors were significantly associated with risk of gastric cancer, including CagA⁺ H. pylori infection (OR, 2.562; 95% CI, 1.174–5.240, P = 0.037), PARP-1 762AA genotype (OR, 1.772; 95% CI, 1.065–3.867; P = 0.042). Stratification analysis indicated that among Cag⁺ H. pylori positive subjects, 762Ala/Ala carriers had higher risk for developing gastric cancer compared with 762Val/Val carrier (OR, 2.337; 95% CI, 1.148–4.758; P = 0.017). Conclusion PARP-1 762Ala/Ala could be a risk factor for gastric cancer in Han Chinese population; PARP-1 762Val/Ala polymorphism and Cag⁺ H. pylori infection jointly contribute to higher risk for gastric cancer.     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

N-acetylcysteine prevents the development of gastritis induced by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> infection

Helicobacter pylori (H. pylori) is a human gastric pathogen, causing various gastric diseases ranging from gastritis to gastric adenocarcinoma. It has been reported that combining N-acetylcysteine (NAC) with conventional antibiotic therapy increases the success rate of H. pylori eradication. We evaluated the effect of NAC itself on the growth and colonization of H. pylori, and development of gastritis, using in vitro liquid culture system and in vivo animal models. H. pylori growth was evaluated in broth culture containing NAC. The H. pylori load and histopathological scores of stomachs were measured in Mongolian gerbils infected with H. pylori strain 7.13, and fed with NAC-containing diet. In liquid culture, NAC inhibited H. pylori growth in a concentration-dependent manner. In the animal model, 3-day administration of NAC after 1 week from infection reduced the H. pylori load; 6-week administration of NAC after 1 week from infection prevented the development of gastritis and reduced H. pylori colonization. However, no reduction in the bacterial load or degree of gastritis was observed with a 6-week administration of NAC following 6-week infection period. Our results indicate that NAC may exert a beneficial effect on reduction of bacterial colonization, and prevents the development of severe inflammation, in people with initial asymptomatic or mild H. pylori infection.     

Lymphocyte proliferative response to <Emphasis Type="Italic">Helicobacter pylori</Emphasis> antigens in <Emphasis Type="Italic">H. pylori</Emphasis>-infected patients

Helicobacter pylori (Hp) contributes to the development of gastric and extra-gastric diseases such as autoimmune thyroiditis (AT), and causes persistent life-long infection despite local and systemic immune response. We determined the specific cellular immune response to Hp antigens and PWM (control mitogen) in two groups of Hp infected patients - group A (n = 21), involving patients with autoimmune thyroiditis and group B (n = 13) of patients without AT - using modified lymphocyte transformation test before and after eradication therapy in comparison with healthy controls (group C, n = 15). Immune reactivity to the majority of Hp antigens (aHp, hHp, HpAg, CagA) was significantly lower in group B before eradication therapy in comparison with healthy Hp negative controls. A significant increase in immune reactivity was observed in group B to certain Hp antigens after successful eradication. The same levels (but insignificant) of immune reactivity were shown in group A. Our results indicate that Hp can cause the inhibition of the specific cellular immune response in Hp infected patients with or without autoimmune diseases such as AT, which can be abrogated by successful eradication of Hp. Lymphocyte transformation test appears to be a good tool for detection of immune memory cellular response in patients with Hp infection.     

Signature of positive selection of <Emphasis Type="Italic">PTK6</Emphasis> gene in East Asian populations: a cross talk for <Emphasis Type="Italic">Helicobacter pylori</Emphasis> invasion and gastric cancer endemicity

Analysis of natural selection events is an attractive strategy for identification of functional variants shaped by gene–environmental interactions and human adaptation. Here, we identified PTK6, a Src-related tyrosine kinase gene, underlying positive selection in East Asian populations. Interestingly, PTK6 variant showed significant correlation with gastric cancer incidences which was the highest in East Asian populations. The high prevalence of gastric cancer in East Asians was also believed to be strongly affected by Helicobacter pylori infection and dietary habit. Therefore, we speculated a competitive interaction of cancer-associated molecules for activation/reduction, where PTK6 likely plays a role through CagA-driven signaling pathway after H. pylori infection. This hypothesis was also supported by our gene expression analysis and the dating of the selective event which was estimated to be ~16,500 years ago, much later than H. pylori invasion in human 50,000 years ago. Establishment of cross talk between PTK6 and CagA by functional studies may further elucidate the underlying biology of H. pylori-mediated gastric cancer.     

Higher number of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> CagA EPIYA C phosphorylation sites increases the risk of gastric cancer,but not duodenal ulcer

Background  

Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric cancer and peptic ulcer. Bacterial virulence factors such as CagA have been shown to increase the risk of both diseases. Studies have suggested a causal role for CagA EPIYA polymorphisms in gastric carcinogenesis, and it has been shown to be geographically diverse. We studied associations between H. pylori CagA EPIYA patterns and gastric cancer and duodenal ulcer, in an ethnically admixed Western population from Brazil. CagA EPIYA was determined by PCR and confirmed by sequencing. A total of 436 patients were included, being 188 with gastric cancer, 112 with duodenal ulcer and 136 with gastritis.     

Frequency of antibiotic resistance in <Emphasis Type="Italic">Helicobacter pylori</Emphasis> strains isolated from the northern population of Iran

The purpose of this study was to evaluate the primary resistance rates of recent clinical Helicobacter pylori isolates to the most commonly used antibiotics in Iran. Two hundreds and ten patients presenting with gastric maladies between January and July of 2009 were enrolled in this study. Endoscopy was performed, and biopsy specimens were collected from each patient for subsequent bacterial culture of H. pylori. Single colony isolates from each patient were then used for antimicrobial susceptibility testing. The disk diffusion method was used to determine susceptibility patterns. One hundred and ninety-seven of the patients were H. pylori positive (93.8%). The rates of resistance to tetracycline, amoxicillin, ciprofloxacin, metronidazole, clarithromycin, and furizoladone were 37.1%, 23.9%, 34.5%, 65.5%, 45.2%, and 61.4%, respectively. A significant association between amoxicillin resistance and disease state (P<0.05) was identified. Furthermore, some double, triple, quadruple, and quintuple combinations of antibiotic resistance were found to be associated with disease state. This study evaluated the prevalence of H. pylori resistance to the most commonly prescribed antibiotics used in Iran and showed that resistance rates were generally higher than previously reported. This data adds to the growing body of evidence that suggests there is increasing antibiotic resistance among H. pylori isolates, which likely is responsible for the decreasing efficacy of anti-H. pylori therapy at the local and global level. Hence, there is a need for continued monitoring of resistance patterns, especially at the local level, and for incorporation of that information into treatment regimens for H. pylori infections.     

Anti-<Emphasis Type="Italic">Helicobacter pylori</Emphasis> effect of mucoadhesive nanoparticles bearing amoxicillin in experimental gerbils model

The purpose of the present study was to design mucoadhesive gliadin nanoparticles (GNP) containing amoxicillin and to evaluate their effectiveness in eradicating Helicobacter pylori. GNP-bearing amoxicillin (AGNP) was prepared by desolvation method. The effect of process variables such as gliadin concentration and initial drug loading on particle size, shape, percent payload, percent entrapment efficiency, in vitro release profile, and mucoadhesive property of GNP was assessed. Rhodamine isothiocyanate-entrapped GNP formulations were prepared to evaluate their in vivo gastric mucoadhesive property in albino rats. With increasing gliadin concentration, the mucoadhesive property of GNP increased. Typically, the maximum amount of nanoparticles remaining was 82±4%, which represented a stronger mucoadhesive propensity and specificity of GNP toward the stomach. In vitro antimicrobial activity of AGNP was evaluated by growth inhibition studies on an isolated H pylori strain. The time required for complete eradication was higher in AGNP than in amoxicillin because of the controlled drug delivery of amoxicillin from AGNP. In vivo clearance of H pylori following oral administration of AGNP to infected Mongolian gerbils was examined. Amoxicillin and AGNP both showed anti-H pylori effects in this experimental model of infection, but the required dose for complete eradication was less in AGNP than in amoxicillin. In conclusion, AGNP eradicated H pylori from the gastrointestinal tract more effectively than amoxicillin because of the prolonged gastrointestinal residence time attributed to mucoadhesion. A dosage form containing mucoadhesive nanoparticles bearing a potential antibiotic should be useful for the complete eradication of H pylori.     

Analysis of the Survival of <Emphasis Type="Italic">H. pylori</Emphasis> Within a Laboratory-based Aquatic Model System Using Molecular and Classical Techniques

Despite the significance of Helicobacter pylori infection for man, its transmission is not clearly known. The human stomach is considered the reservoir of this pathogen, and one of the accepted routes is fecal–oral, in which water acts as a vector. However, although H. pylori epidemiology associates its transmission with water, only molecular and not cultural analysis detects the bacteria in water. This study was carried out to understand these data through studying the survival of H. pylori in a laboratory water model using cultural, morphological, and molecular methods. A mineral water system spiked with H. pylori and stored at 7 ± 1°C in the dark was analyzed by different methods over a period of 3 weeks. The total number of cells observed by DAPI staining and their DNA content remained constant over this study period. In contrast, cells could no longer be cultured after 5 days. Cell viability, which was determined via the LIVE/DEAD BacLight kit, decreased up to day 14, and at day 21 all cell membranes were damaged. In addition, a gradual conversion from spiral to coccal morphology occurred from day 3 onward. However, polymerase chain reaction (PCR) technique detected H. pylori DNA at day 21 and 3 months later. A study of the cell morphology of a young colony demonstrated the coexistence of bacilli and cocci. The results of this study show that H. pylori survives in water but loses its culturability and bacillar morphology rapidly, although it remains viable for longer periods and its DNA is still detectable much later. Thus, interpreting H. pylori‘s behavior in water differs according to the type of analysis. Consequently, we suggest that the presence of H. pylori infective cells is overestimated by PCR, whereas, in contrast, culture techniques underestimate it. Nevertheless, H. pylori should be considered a waterborne pathogen during its viable period, independently of its shape and culturability, as its presence in water may be risky for human health.     

Surface expression of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> urease subunit B gene E fragment on <Emphasis Type="Italic">Lactococcus lactis</Emphasis> by means of the cell wall anchor of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> protein A

Fragment E of ureB (ureBE) was cloned from a clinical isolate of Helicobacter pylori. A prokaryotic expression vector, pAMJ399, with the ureB fragment E and the Staphylococcus aureus protein A anchor fragment (spaX), was constructed. The fusion protein was expressed under the control of the P170 promoter in Lactococcus lactis. Western blot assay of lactococcal cell wall extracts with a polyclonal chicken antiserum confirmed the immunity of the expressed recombinant protein which was located on the cell surface. These results provide the first report of a surface display system in lactic acid bacteria for the delivery of oral vaccines against Helicobacter pylori.