Development of a selective medium for isolation of Helicobacter pylori from cattle and beef samples

Helicobacter pylori has been isolated from the human stomach with media containing only minimal selective agents. However, current research on the transmission and sources of infection requires more selective media due to the higher numbers of contaminants in environmental, oral, and fecal samples. The objective of this study was to develop and evaluate detection techniques that are sufficiently selective to isolate H. pylori from potential animal and food sources. Since H. pylori survives in the acidic environment of the stomach, low pH with added urea was studied as a potential selective combination. H. pylori grew fairly well on H. pylori Special Peptone plating medium supplemented with 10 mM urea at pH 4. 5, but this pH did not sufficiently inhibit the growth of contaminants. Various antibiotic combinations were then compared, and a combination consisting of 10 mg of vancomycin per liter, 5 mg of amphotericin B per liter, 10 mg of cefsulodin per liter, 62,000 IU of polymyxin B sulfate per liter, 40 mg of trimethoprim per liter, and 20 mg of sulfamethoxazole per liter proved to be highly selective but still allowed robust colonies of H. pylori to grow. This medium was highly selective for recovering H. pylori from cattle and beef samples, and it is possible that it could be used to enhance the recovery of this bacterium from human and environmental samples, which may be contaminated with large numbers of competing microorganisms.     

Development of an Internal Control for Evaluation and Standardization of a Quantitative PCR Assay for Detection of Helicobacter pylori in Drinking Water

Due to metabolic and morphological changes that can prevent Helicobacter pylori cells in water from growing on conventional media, an H. pylori-specific TaqMan quantitative PCR (qPCR) assay was developed that uses a 6-carboxyfluorescein-labeled probe (A. E. McDaniels, L. Wymer, C. Rankin, and R. Haugland, Water Res. 39:4808-4816, 2005). However, proper internal controls are needed to provide an accurate estimate of low numbers of H. pylori in drinking water. In this study, the 135-bp amplicon described by McDaniels et al. was modified at the probe binding region, using PCR mutagenesis. The fragment was incorporated into a single-copy plasmid to serve as a PCR-positive control and cloned into Escherichia coli to serve as a matrix spike. It was shown to have a detection limit of five copies, using a VIC dye-labeled probe. A DNA extraction kit was optimized that allowed sampling of an entire liter of water. Water samples spiked with the recombinant E. coli cells were shown to behave like H. pylori cells in the qPCR assay. The recombinant E. coli cells were optimized to be used at 10 cells/liter of water, where they were shown not to compete with 5 to 3,000 cells of H. pylori in a duplex qPCR assay. Four treated drinking water samples spiked with H. pylori (100 cells) demonstrated similar cycle threshold values if the chlorine disinfectant was first neutralized by sodium thiosulfate.     

Helicobacter pylori comb. nov. Exhibits Facultative Acidophilism and Obligate Microaerophilism

Modified brucella broth medium was used to study the growth of Helicobacter pylori at varied pHs and partial pressures of oxygen and to determine the effect of urea on culture pH. Our findings suggested that the pHs of the media remained stable with or without urea and that H. pylori showed facultative acidophilism and obligate microaerophilism.     

Evaluation of Culture Techniques for Isolation of Pseudomonas pseudomallei from Soil

Three selective enrichment broths and four selective agar media were evaluated for their ability to support the growth of Pseudomonas pseudomallei both at 35°C and at ambient temperature (range, 20 to 32°C; mean, 25°C). Colony counts of 50 strains of P. pseudomallei and recovery studies with 1 soil strain in 60 simulated soil samples demonstrated that enrichment with Trypticase soy broth incorporating 5 mg of crystal violet per liter and 20 mg of colistin per liter (CVCB) and subculture to Ashdown medium supported the growth of all 50 strains and produced the highest recovery rates with the greatest suppression of other soil flora. An enrichment broth of MacConkey broth (purple) incorporating 10 mg of crystal violet per liter, 5 mg of bromcresol purple per liter, 25 mg of gentamicin per liter, and 650 mg of streptomycin per liter showed greater suppression of soil bacteria than CVCB, but it failed to support the growth of three strains of P. pseudomallei. Recovery rates were essentially the same irrespective of whether the soil samples were incubated at 35°C or at ambient temperature, provided cultures were incubated in protected shade for an extended period. This is an important feature for field work in large-scale epidemiological surveys in which resources are limited.     

Development of a Plating Medium for Selection of Helicobacter pylori from Water Samples

The goal of this study was to develop a simple plating medium to allow large-scale screening of water samples for the presence of Helicobacter pylori. Five conventional plating media (brain heart infusion, brucella agar, Columbia blood agar base, campylobacter agar kit Skirrow, and HPSPA medium), each containing a commercial antibiotic supplement, were initially evaluated. Eight strains selected as common waterborne organisms (Acinetobacter, Aeromonas, Bacillus, Escherichia coli, Enterobacter, Enterococcus, Helicobacter pylori, and Pseudomonas strains) were individually plated onto each of these media. Three organisms (Acinetobacter, E. coli, and H. pylori) were able to grow on all five media. This growth was unacceptable since Helicobacter grows very slowly and competing organisms must be inhibited for up to 7 days. Therefore, a more selective medium (HP agar) containing a novel mixture of growth supplements plus amphotericin B and polymyxin B was developed. This medium also included a phenol red color indicator for urease production. Aliquots of nonsterile well water that contained native flora (Flavobacterium, Serratia, Citrobacter, Pasteurella, Ochrobactrum, Rahnella, and unidentified molds) and were further adulterated with the eight strains listed above (10⁶ CFU of each strain per 100 ml) were spiked with H. pylori and were plated. In spite of the heavy mixed microbial load, only H. pylori colonies grew during 7 days of incubation at 37°C. The color indicator system allowed presumptive identification of H. pylori colonies sooner (12 to 20 h) than the conventional media tested allowed. The HP formulation developed in this study provides a medium with superior selectivity for H. pylori from mixed microbial populations in water and reduces the time required to complete the assay.     

Effect of Chlorine on Giardia lamblia Cyst Viability

The effect of chlorine concentration on Giardia lamblia cyst viability was tested under a variety of conditions. The ability of Giardia cysts to undergo excystation was used as the criterion of viability. The experimental variables employed included temperature (25, 15, and 5°C), pH (6, 7, and 8), chlorine-cyst contact time (10, 30, and 60 min), and chlorine concentration (1 to 8 mg/liter). In the pH range studied, cyst survival generally was observed to increase as buffer pH increased. Water temperature coupled with chlorination proved to be important in cyst survival. Results of these experiments at the three temperatures studied can be summarized as follows: at 25°C, exposure to 1.5 mg/liter for 10 min killed all cysts at pH 6, 7, and 8. At 15°C, 2.5 mg of chlorine per liter for 10 min killed all cysts at pH 6, but at pH 7 and 8 small numbers of cysts remained viable after 30 min but not after 60 min. At 5°C, 1 mg of chlorine per liter for 60 min failed to kill all the cysts at any pH tested. At this temperature, 2 mg of chlorine per liter killed all cysts after 60 min at pH 6 and 7, but not at pH 8. A chlorine concentration of 4 mg/liter killed all the cysts at all three pH values after 60 min, but not after 30 min. A chlorine concentration of 8 mg/liter killed all Giardia cysts at pH 6 and 7 after contact for 10 min, and at pH 8 after 30 min. This study points up the role of temperature, pH, and chlorine demand in the halogen treatment of drinking water to destroy cysts. It also raises an epidemiological problem, namely: low water temperatures, where killing of Giardia requires relatively high chlorine concentrations and long contact times, are (i) to be expected in many areas where epidemic waterborne giardiasis has been reported and (ii) particularly conducive to the long-term survival of Giardia cysts.     

Characterization of Helicobacter pylori Bacteriophage KHP30

Helicobacter pylori inhabits the stomach mucosa and is a causative agent of stomach ulcer and cancer. In general, bacteriophages (phages) are strongly associated with bacterial evolution, including the development of pathogenicity. Several tailed phages have so far been reported in H. pylori. We have isolated an H. pylori phage, KHP30, and reported its genomic sequence. In this study, we examined the biological characteristics of phage KHP30. Phage KHP30 was found to be a spherical lipid-containing phage with a diameter of ca. 69 nm. Interestingly, it was stable from pH 2.5 to pH 10, suggesting that it is adapted to the highly acidic environment of the human stomach. Phage KHP30 multiplied on 63.6% of clinical H. pylori isolates. The latent period was ca. 140 min, shorter than the doubling time of H. pylori (ca. 180 min). The burst size was ca. 13, which was smaller than the burst sizes of other known tailed or spherical phages. Phage KHP30 seemed to be maintained as an episome in H. pylori strain NY43 cells, despite a predicted integrase gene in the KHP30 genomic sequence. Seven possible virion proteins of phage KHP30 were analyzed using N-terminal protein sequencing and mass spectrometry, and their genes were found to be located on its genomic DNA. The genomic organization of phage KHP30 differed from the genomic organizations in the known spherical phage families Corticoviridae and Tectiviridae. This evidence suggests that phage KHP30 is a new type of spherical phage that cannot be classified in any existing virus category.     

Comparison of Media for the Isolation of Cryptococcus neoformans

Four media, Staib's Guizotia abyssinica, trypan blue, and Staib's with 2 and 10 mg of methyl violet per liter, were compared for the selective and differential isolation of Cryptococcus neoformans from environmental samples. Trypan blue medium allowed for the differentiation of C. neoformans colonies from Candida albicans colonies several days earlier than did Staib's medium. The addition of methyl violet to Staib's medium was found to be inhibitory to some strains of all species tested. Diphenyl in Staib's medium inhibited the growth of 30 strains of C. neoformans and C. albicans.     

Structure of the human gastric bacterial community in relation to Helicobacter pylori status

The human stomach is naturally colonized by Helicobacter pylori, which, when present, dominates the gastric bacterial community. In this study, we aimed to characterize the structure of the bacterial community in the stomach of patients of differing H. pylori status. We used a high-density 16S rRNA gene microarray (PhyloChip, Affymetrix, Inc.) to hybridize 16S rRNA gene amplicons from gastric biopsy DNA of 10 rural Amerindian patients from Amazonas, Venezuela, and of two immigrants to the United States (from South Asia and Africa, respectively). H. pylori status was determined by PCR amplification of H. pylori glmM from gastric biopsy samples. Of the 12 patients, 8 (6 of the 10 Amerindians and the 2 non-Amerindians) were H. pylori glmM positive. Regardless of H. pylori status, the PhyloChip detected Helicobacteriaceae DNA in all patients, although with lower relative abundance in patients who were glmM negative. The G2-chip taxonomy analysis of PhyloChip data indicated the presence of 44 bacterial phyla (of which 16 are unclassified by the Taxonomic Outline of the Bacteria and Archaea taxonomy) in a highly uneven community dominated by only four phyla: Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Positive H. pylori status was associated with increased relative abundance of non-Helicobacter bacteria from the Proteobacteria, Spirochetes and Acidobacteria, and with decreased abundance of Actinobacteria, Bacteroidetes and Firmicutes. The PhyloChip detected richness of low abundance phyla, and showed marked differences in the structure of the gastric bacterial community according to H. pylori status.     

Antagonistic Activity against Helicobacter Infection In Vitro and In Vivo by the Human Lactobacillus acidophilus Strain LB

The purpose of the present study was to examine the activity of the human Lactobacillus acidophilus strain LB, which secretes an antibacterial substance(s) against Helicobacter pylori in vitro and in vivo. The spent culture supernatant (SCS) of the strain LB (LB-SCS) dramatically decreased the viability of H. pylori in vitro independent of pH and lactic acid levels. Adhesion of H. pylori to the cultured human mucosecreting HT29-MTX cells decreased in parallel with the viability of H. pylori. In conventional mice, oral treatment with the LB-SCS protected against infection with Helicobacter felis. Indeed, at both 8 and 49 days post-LB-SCS treatment (29 and 70 days postinfection), inhibition of stomach colonization by H. felis was observed, and no evidence of gastric histopathological lesions was found. LB-SCS treatment inhibits the H. pylori urease activity in vitro and in H. pylori that remained associated with the cultured human mucosecreting HT29-MTX cells. Moreover, a decrease in urease activity was detected in the stomach of the mice infected with H. felis and treated with LB-SCS.     

Effect of Urea Concentration on Growth of Ureaplasma urealyticum (T-Strain Mycoplasma)

The effect of urea on growth of Ureaplasma urealyticum type VIII was studied by cultivating the organisms in a dialysate broth, prepared from soy peptone and autoclaved yeast, supplemented with 5% dialyzed horse serum, 100 mM 2-(N-morpholino)ethane sulfonic acid buffer (pH 5.75), and defined amounts of urea. Without urea, growth did not occur. Total growth was directly related to urea concentration. The least amount of urea that supported growth was 0.032 mM, which resulted in 3 × 10⁴ colony-forming units per ml. The maximum yield of organisms, 8.0 × 10⁷ colony-forming units per ml, was observed at 32 mM urea. Growth was limited not only by urea concentration, but also by the buffer capacity of the medium. The maximum amount of 2-(N-morpholino)ethane sulfonic acid buffer that could be employed was 100 mM; at higher concentrations, growth was inhibited. The yield of U. urealyticum was small even in medium with 32 mM urea and 100 mM 2-(N-morpholino)ethane sulfonic acid buffer: 0.63 mg of protein per liter of culture containing 5 × 10¹⁰ total colony-forming units. The molar growth yield was 20 mg of protein per mol of urea. The growth rate was also a function of urea concentration. Generation times ranged from 8 h at 0.032 mM urea to 1.6 h at 3.2 mM urea, where the substrate level was saturating. The K_s value for growth was 2.0 × 10⁻⁴ M urea. Thus, urea is a growth-limiting factor for U. urealyticum, but remarkably large amounts of this substrate are required.     

Detection of Hemolysin Variants of Shiga Toxin-Producing Escherichia coli by PCR and Culture on VancomycinCefixime-Cefsulodin Blood Agar

The presence of a hemolysin-encoding gene, elyA or hlyA, from Shiga toxin-producing Escherichia coli (STEC) was detected by PCR in each of 95 strains tested. PCR products of elyA from human STEC isolates of serovars frequently detected in Germany, such as O157:H−, O103:H2, O103:H−, O26:H11, and O26:H−, showed nucleotide sequences identical to previously reported ones for O157:H7 and O111:H− strains. Compared to them, four elyA amplicons derived from human isolates of rare STEC serovars showed identity of about 98% but lacked an AluI restriction site. However, the nucleotide sequence of an amplicon derived from a porcine O138:K81:H− STEC strain was identical to the corresponding region of hlyA, encoding alpha-hemolysin, from E. coli. This hlyA amplicon showed 68% identity with the nucleotide sequence of the corresponding elyA fragment. It differed from the elyA PCR product in restriction fragments generated by AluI, EcoRI, and MluI. Of the 95 representative STEC strains, 88 produced hemolysin on blood agar supplemented with vancomycin (30 mg/liter), cefixime (20 μg/liter), and cefsulodin (3 mg/liter) (BVCC). The lowest added numbers of two to six STEC CFU per g of stool or per ml of raw milk were detectable on BVCC plates after seeding of the preenrichment broth, modified tryptic soy broth (mTSB) supplemented with novobiocin (10 mg/liter), with 16 STEC strains. These strains represented the seven prevailing serovars diagnosed from German patients. However, with ground-beef samples, PCR was essential to identify the lowest added numbers of two to six STEC CFU among colonies of hemolyzing Enterobacteriaceae, such as Serratia spp. and alpha-hemolysin-producing E. coli. We conclude that preenrichment of stool and food samples in mTSB for 6 h followed by overnight culturing on BVCC is a simple method for the isolation and presumptive identification of STEC.     

Evaluation of a four-drug, three-antibiotic, nonbismuth-containing "concomitant" therapy as first-line Helicobacter pylori eradication regimen in Greece

Background: The eradication rates of Helicobacter pylori (H. pylori) with standard treatments are decreasing worldwide as in Greece. Studies with new antibiotic combinations are needed to find better methods of eradication. Therefore, the aim of this study was to evaluate efficacy and tolerability of a 10‐day, four‐drug, three‐antibiotic, nonbismuth–containing concomitant regimen. Materials and Methods: This is a prospective, open‐label, multicenter study that included 131 patients infected with H. pylori. All patients were diagnosed with peptic ulcer disease or nonulcer dyspepsia by endoscopy. H. pylori infection was established by at least two positive tests among rapid urease test, gastric histology, and ¹³C‐urea breath test. For 10 days, all patients received esomeprazole 40 mg, amoxycillin 1000 mg, clarithromycin 500 mg, and metronidazole 500 mg, all b.d. eradication was assessed with ¹³C urea breath test 8 weeks after the start of treatment. Intention‐to‐treat and per‐protocol eradication rates were determined. Results: One hundred and twenty‐seven of the 131 patients completed the study. At intention‐to‐treat analysis, the eradication rate was 91.6% (95% confidence interval (CI), 85.5–95.7%). For the per‐protocol analysis, the eradication rate was 94.5% (95% CI, 89–97.8%). Adverse events were noted in 42 of 131 (32.1%); drug compliance was excellent with 96.9% of the patients taking more than 90% of the prescribed medication. Conclusion: A 10‐day concomitant regimen appears to be an effective, safe, and well‐tolerated treatment option for first‐line H. pylori eradication in Greece.     

Gentamicin-Based Medium for the Isolation of Group D Streptococci and Application of the Medium to Water Analysis

Gentamicin-thallous-carbonate (GTC) medium contained (per liter): 40.0 g of Trypticase soy agar, 5.0 g of KH₂PO₄, 2.0 g of NaHCO₂, 1.0 g of glucose, 1.0 g of esculin, 0.5 g of thallous acetate (TA), 0.5 g of ferric citrate, 0.75 ml of Tween 80, and 2.5 mg of gentamicin sulfate. The NaHCO₃ (20 ml of a 10% solution that had been heated to boiling) was added after sterilization of the basal medium. The spread plate technique was used to compare GTC agar with Pfizer selective enterococcus, TA, and KF agars by using sewage as well as bovine and swine fecal samples. Significantly greater numbers of group D streptococci were recovered on GTC agar than on Pfizer selective enterococcus or KF agars, within and over all samples. Higher counts also were obtained on GTC than on TA agar, but the differences were not statistically significant. The percentage of false positives was about the same for all four media. Samples of riverwater also were plated on GTC, TA, and KF agars, and significantly higher recoveries were obtained with GTC agar. GTC agar was superior to the other media examined primarily because of increased recoveries of Streptococcus bovis and S. equinus; other advantages of GTC agar were large colony size and short (24-h) incubation period. The percentage of false positives from riverwater was 13% for GTC agar and 0% for TA and KF agars; therefore, confirmation would be necessary when GTC agar is used with some types of environmental samples.     

Factors Affecting Yield and Safety of Protein Production from Cassava by Cephalosporium eichhorniae

The properties of Cephalosporium eichhorniae 152 (ATCC 38255) affecting protein production from cassava carbohydrate, for use as an animal feed, were studied. This strain is a true thermophile, showing optimum growth at 45° to 47°C, maximum protein yield at 45°C, and no growth at 25°C. It has an optimum pH of about 3.8 and is obligately acidophilic, being unable to sustain growth at pH 6.0 and above in a liquid medium, or pH 7.0 and above on solid media. The optimum growth conditions of pH 3.8 and 45°C were strongly inhibitive to potential contaminants. It rapidly hydrolyzed cassava starch. It did not utilize sucrose, but some (around 16%) of the small sucrose component of cassava was chemically hydrolyzed during the process. Growth with cassava meal (50 g/liter [circa 45 g/liter, glucose equivalent]) was complete in around 20 h, yielding around 22.5 g/liter (dry biomass), containing 41% crude protein (48 to 50% crude protein in the mycelium) and 31% true protein (7.0 g/liter). Resting and germinating spores (10⁶ to 10⁸ per animal) injected by various routes into normal and γ-irradiated 6-week-old mice and 7-day-old chickens failed to initiate infections.     

Quantitation of Helicobacter pylori ureC gene and its comparison with different diagnostic techniques and gastric histopathology

Numerous diagnostic assays for Helicobacter pylori detection are available. However, these techniques have their own advantages as well as limitations. Here we tried to develop a real-time quantitative (Q) PCR assay to measure ureC copy number to detect H. pylori, based on the fact that there is only one copy of the ureC gene per bacterium. We enrolled 120 adult patients [non-ulcer dyspepsia (NUD) 60, peptic ulcer disease (PUD) 20, gastric cancer (GC) 40] undergoing upper gastrointestinal endoscopies. During each endoscopic examination, antral biopsies from normal region of the antrum were obtained and subjected to the following tests: RUT, culture, histopathology, H. pylori-specific ureC PCR and ureC Q-PCR. Calculation of H. pylori copy number was based on the standard curve generated using 10-fold dilutions of DNA extracted from the H. pylori control strain varying from 10⁵ to 10¹ copies. The prevalence of H. pylori infection in our study population was 54% with no significant difference among disease and control population. The sensitivity of Q-PCR was found to be 100% which was highest among all diagnostic tests. The established Q-PCR is around 10 times more sensitive than the conventional PCR method. The copy number of H. pylori DNA was significantly increased when overall gastritis, H. pylori density, chronic inflammation and intestinal metaplasia were present. In summary, we developed a rapid and sensitive Q-PCR method for detecting H. pylori. This technique offers a significant improvement over other available methods for detecting H. pylori in clinical and research samples.     

Human Immune Responses to H. pylori HLA Class II Epitopes Identified by Immunoinformatic Methods

H. pylori persists in the human stomach over decades and promotes several adverse clinical sequelae including gastritis, peptic ulcers and gastric cancer that are linked to the induction and subsequent evasion of chronic gastric inflammation. Emerging evidence indicates that H. pylori infection may also protect against asthma and some other immune-mediated conditions through regulatory T cell effects outside the stomach. To characterize the complexity of the CD4+ T cell response generated during H. pylori infection, computational methods were previously used to generate a panel of 90 predicted epitopes conserved among H. pylori genomes that broadly cover HLA Class II diversity for maximum population coverage. Here, these sequences were tested individually for their ability to induce in vitro responses in peripheral blood mononuclear cells by interferon-γ ELISpot assay. The average number of spot-forming cells/million PBMCs was significantly elevated in H. pylori-infected subjects over uninfected persons. Ten of the 90 peptides stimulated IFN-γ secretion in the H. pylori-infected group only, whereas two out of the 90 peptides elicited a detectable IFN-γ response in the H. pylori-uninfected subjects but no response in the H. pylori-infected group. Cytokine ELISA measurements performed using in vitro PBMC culture supernatants demonstrated significantly higher levels of TNF-α, IL-2, IL-4, IL-6, IL-10, and TGF-β1 in the H. pylori-infected subjects, whereas IL-17A expression was not related to the subjects H. pylori-infection status. Our results indicate that the human T cell responses to these 90 peptides are generally increased in actively H. pylori-infected, compared with H. pylori-naïve, subjects. This information will improve understanding of the complex immune response to H. pylori, aiding rational epitope-driven vaccine design as well as helping identify other H. pylori epitopes with potentially immunoregulatory effects.     

Helicobacter pylori's Unconventional Role in Health and Disease

The discovery of a bacterium, Helicobacter pylori, that is resident in the human stomach and causes chronic disease (peptic ulcer and gastric cancer) was radical on many levels. Whereas the mouth and the colon were both known to host a large number of microorganisms, collectively referred to as the microbiome, the stomach was thought to be a virtual Sahara desert for microbes because of its high acidity. We now know that H. pylori is one of many species of bacteria that live in the stomach, although H. pylori seems to dominate this community. H. pylori does not behave as a classical bacterial pathogen: disease is not solely mediated by production of toxins, although certain H. pylori genes, including those that encode exotoxins, increase the risk of disease development. Instead, disease seems to result from a complex interaction between the bacterium, the host, and the environment. Furthermore, H. pylori was the first bacterium observed to behave as a carcinogen. The innate and adaptive immune defenses of the host, combined with factors in the environment of the stomach, apparently drive a continuously high rate of genomic variation in H. pylori. Studies of this genetic diversity in strains isolated from various locations across the globe show that H. pylori has coevolved with humans throughout our history. This long association has given rise not only to disease, but also to possible protective effects, particularly with respect to diseases of the esophagus. Given this complex relationship with human health, eradication of H. pylori in nonsymptomatic individuals may not be the best course of action. The story of H. pylori teaches us to look more deeply at our resident microbiome and the complexity of its interactions, both in this complex population and within our own tissues, to gain a better understanding of health and disease.Common wisdom circa 1980 suggested that the stomach, with its low pH, was a sterile environment. Then, endoscopy of the stomach became common and, in 1984, pathologist Robin Warren and gastroenterologist Barry Marshall saw an extracellular, curved bacillus, often in dense sheets, lining the stomach epithelium of patients with gastritis (inflammation of the stomach) and ulcer disease [1]. Soon, the medical community understood that the gram-negative bacterium Helicobacter pylori, not stress, is the major cause of stomach inflammation, which, in some infected individuals, precedes peptic ulcer disease (10%–20%), distal gastric adenocarcinoma (1%–2%), and gastric mucosal-associated lymphoid tissue (MALT) lymphoma (<1%) [2]–[5]. Thus, H. pylori gained distinction as the only known bacterial carcinogen [6]. It is believed that half of the world''s population is infected with H. pylori; however, the burden of disease falls disproportionately on less-developed countries. The incidence of infection in developed countries has fallen dramatically, for unknown reasons, with a corresponding decrease in gastric cancer [7]. This public health success is tempered by the recent demonstration of an inverse relationship between H. pylori infection and esophageal adenocarcinoma, Barrett''s esophagus, and reflux esophagitis [8]. H. pylori has been with humans since our earliest days, thus it is not surprising that its relationship is that of both a commensal bacterium and a pathogen, causing some diseases and possibly protecting against others. In addition, it is genetically diverse, likely as a result of constant exposure to both environmental and immunological selection, suggesting that genetic diversification is a strategy for long-term colonization.     

Growth Inhibitory,Bactericidal, and Morphostructural Effects of Dehydrocostus Lactone from Magnolia sieboldii Leaves on Antibiotic-Susceptible and -Resistant Strains of Helicobacter pylori

Helicobacter pylori is associated with various diseases of the upper gastrointestinal tract, such as gastric inflammation and duodenal and gastric ulcers. The aim of the study was to assess anti-H. pylori effects of the sesquiterpene lactone dehydrocostus lactone (DCL) from Magnolia sieboldii leaves, compared to commercial pure DCL, two previously known sesquiterpene lactones (costunolide and parthenolide), (–)-epigallocatechin gallate, and four antibiotics. The antibacterial activity of natural DCL toward antibiotic-susceptible H. pylori ATCC 700392 and H. pylori ATCC 700824 strains (MIC, 4.9 and 4.4 mg/L) was similar to that of commercial DCL and was more effective than costunolide, parthenolide, and EGCG. The activity of DCL was slightly lower than that of metronidazole (MIC, 1.10 and 1.07 mg/L). The antibacterial activity of DCL was virtually identical toward susceptible and resistant strains, even though resistance to amoxicillin (MIC, 11.1 mg/L for PED 503G strain), clarithromycin (49.8 mg/L for PED 3582GA strain), metronidazole (21.6 mg/L for H. pylori ATCC 43504 strain; 71.1 mg/L for 221 strain), or tetracycline (14.2 mg/L for B strain) was observed. This finding indicates that DCL and the antibiotics do not share a common mode of action. The bactericidal activity of DCL toward H. pylori ATCC 43504 was not affected by pH values examined (4.0–7.0). DCL caused considerable conversion to coccoid form (94 versus 49% at 8 and 4 mg/L of DCL for 48 h). The Western blot analysis revealed that urease subunits (UreA and UreB) of H. pylori ATCC 43504 were not affected by 10 mM of DCL, whereas UreA monomer band completely disappeared at 0.1 mM of (–)-epigallocatechin gallate. Global efforts to reduce the level of antibiotics justify further studies on M. sieboldii leaf-derived materials containing DCL as potential antibacterial products or a lead molecule for the prevention or eradication of drug-resistant H. pylori.     

A protocol for isolating putative Helicobacter pylori from fecal specimens and genotyping using vacA alleles

Background. This study outlines steps for isolating and culturing Helicobacter pylori from freshly voided fecal specimens and genotyping isolates for vacA alleles. Materials and methods. A family with four H. pylori‐infected members participated in this pilot study. Criterion for participation was a positive test for H. pylori by the urea breath test. Fecal specimens from children were taken from a freshly soiled diaper, placed in cold buffer, and prepared for culture in less than 2 hours. Culturing of H. pylori utilized selective culture media and isolates were screened for negative Gram stain, positive catalase and oxidase tests, and positive H. pylori 16S ribosomal RNA polymerase chain reaction (PCR). Strain types were determined by vacA genotyping. Results. The isolation procedure is relatively simple, although 5–7 days are required for H. pylori culturing. Isolation and purification of DNA eliminated PCR inhibitors and resulted in reliable analyses. All four family members were infected with the same H. pylori strain with a genotype of vacA s1a/m2. Conclusion. This research lays the foundation for developing a routine and direct noninvasive method to detect the presence of H. pylori in fecal specimens. It is especially convenient for diagnosing children and infants, as samples can be obtained from soiled diapers. Culturing H. pylori from fecal samples in certain cases is important for antibiotic resistant studies prior to treating infected patients and for strain genotyping in epidemiological studies to determine transmission.