Colony formation by Helicobacter pylori after long-term incubation under anaerobic conditions

To evaluate the viability of Helicobacter pylori cultured under anaerobic conditions, H. pylori strain TK1029 was grown on blood agar in a microaerophilic environment at 37 degrees C for 4 days, and subsequently cultured under anaerobic conditions for 1 to 35 days. Colony formation by bacteria on blood agar plates cultured under anaerobic conditions was observed only for up to 4 days of microaerophilic incubation. By Gram staining, the morphological form of the bacteria was shown to be predominantly coccoid. However, bacteria cultured under anaerobic conditions for 15 to 35 days formed colonies on blood agar after pre-incubation of bacteria with PBS, but not without pre-incubation. These results suggest that H. pylori survives long-term culture under anaerobic conditions and that both pre-incubation in non-nutrient solution and high density of bacterial concentration might be important for recovery of H. pylori cultured for a prolonged time under anaerobic conditions.     

Optimized conditions for the fermentation of Helicobacter pylori and production of vacuolating cytotoxin

Abstract We report here improvements to the growth media and fermentation conditions which result in a substantial increase of Helicobacter pylori growth and in the enhanced production of vacuolating cytotoxin. Addition of glucose to the medium resulted in the increase of cell yield, cell viability and a significant improvement in the production of vacuolating cytotoxin.     

Mucoid Helicobacter pylori isolates with fast growth under microaerobic and aerobic conditions

Background: Helicobacter pylori is microaerobic and turns into coccoid under aerobic conditions. In this study, two mucoid strains, A and D, were isolated from gastric biopsies which grew well on blood agar after 24‐hour incubation under aerobic as well as microaerobic conditions. The aim of this study was to identify these strains and compare their growth under aerobic and microaerobic conditions with that of control H. pylori. Materials and Methods: The two isolates A and D were identified as H. pylori according to microscopic morphology, urease, catalase and oxidase tests. Their growth under humidified aerobic and microaerobic conditions was compared with that of control H. pylori which grew only under microaerobic conditions. They were further identified by amplification of 16S rRNA, vacA alleles, cagA and ureAB genes by PCR. Their susceptibility to current antimicrobials was also examined. Results: The strains A and D produced mucoid colonies under aerobic and microaerobic conditions after 24‐hour, exhibiting the typical spiral morphology of H. pylori. The results of urease, catalase and oxidase tests were positive. Sequencing of amplified products showed 99–100% homology with those of the reference H. pylori strains in GenBank. Both strains exhibited resistance to the high concentrations of antimicrobials. Conclusions: This study reports the isolation of two mucoid strains of H. pylori with confluent growth under aerobic and microaerobic conditions. It appears that production of exopolysaccharide (EXP) could serve as a physical barrier to reduce oxygen diffusion into the bacterial cell and uptake of antibiotics. EXP protected the mucoid H. pylori isolates against stressful conditions, the result of which could be persistence of bacterial infection in the stomach.     

Characterization of arginine transport in Helicobacter pylori

Background. The amino acid L‐arginine is an essential requirement for growth of Helicobacter pylori. Several physiological roles of this amino acid have been identified in the bacterium, but very little is known about the transport of L‐arginine and of other amino acids into H. pylori. Methods. Radioactive tracer techniques using L‐(U‐¹⁴C) arginine and the centrifugation through oil method were employed to measure the kinetic parameters, temperature dependence, substrate specificity, and effects of analogues and inhibitors on L‐arginine transport. Results. The transport of arginine at millimolar concentrations was saturable with a K_m of 2.4 ± 0.3 mM and V_max of 1.3 ± 0.2 pmole min^?1 (µl cell water)^?1 or 31 ± 3 nmole per minute (mg protein)^?1 at 20°C, depended on temperature between 4 and 40°C, and was susceptible to inhibitors. These characteristics suggested the presence of one or more arginine carriers. The substrate specificity of the transport system was studied by measuring the effects of L‐arginine analogues and amino acids on the rates of transport of L‐arginine. The absence of inhibition in competition experiments with L‐lysine and L‐ornithine indicated that the transport system was not of the Lysine‐Arginine‐Ornithine or Arginine‐Ornithine types. The presence of different monovalent cations did not affect the transport rates. Several properties of L‐arginine transport were elucidated by investigating the effects of potential inhibitors. Conclusions. The results provided evidence that the transport of L‐arginine into H. pylori cells was carrier‐mediated transport with the driving force supplied by the chemical gradient of the amino acid.     

Characterization of an Helicobacter pylori environmental strain

Aims:  To investigate the main genotypic virulence markers and the phenotypic features of an environmental Helicobacter pylori strain, named MDC1.
Methods and Results:  The H. pylori MDC1 genotypic status was evaluated by PCR amplification. The mosaicism in vac A alleles was expressed by the s1m1 allelic combination, as found in strains which are strong vacuolating cytotoxin producers; the number of cag A variable EPIYA motifs displayed P1P2P3P3 pattern and the ice A1 was recorded between the ice A allelic types and the bab A2 gene found in strains causing more severe disease. The biofilm formation was evaluated on a polystyrene surface in static conditions by scanning electron microscopy and confocal scanning laser microscopy. Helicobacter pylori MDC1 displayed a dense mature biofilm with cells in a coccoid morphology persistent in time in which the expression of the lux S gene, related to the quorum-sensing signalling, was always detected.
Conclusions:  Helicobacter pylori MDC1 strain had the main virulence markers closely related to gastric pathogenesis and displayed a well-structured biofilm which allowed this bacterium to be more protected in the environment.
Significance and Impact of the Study:  The persistence of the environmental virulent H. pylori strain in a clustered state suggests a long-term survival of this bacterial community outside of the host, enabling the bacterial transmission with important clinical repercussions.     

幽门螺杆菌空泡毒素研究进展

幽门螺杆菌空泡毒素是该菌产生的已知其它细菌毒素无明显源性的唯一蛋白毒素。该毒素是幽门螺杆菌重要的毒力致病因子,它的产生与感染胃肠上皮损伤和溃疡形成密切相关。本就幽门螺杆菌空泡毒素的结构与功能研究进展以及在未来免疫预防与免疫治疗中的作用进行了阐述。     

Adhesion of water stressed Helicobacter pylori to abiotic surfaces

AIM: The main aim of this work was to study and compare the adhesion of water exposed Helicobacter pylori to six different substrata and correlate any changes in morphology, physiology, ability to form aggregates and cultivability when in the planktonic or in the sessile phase. METHODS AND RESULTS: The number of total cells adhered for different water exposure times and modifications in the cell shape were evaluated using epifluorescence and scanning electron microscopy, and physiology assessed using Syto9 and propidium iodide (PI) cellular uptake. All abiotic surfaces were rapidly colonized by H. pylori, and colonization appeared to reach a steady state after 96 h with levels ranging from 2.3 x 10(6) to 3.6 x 10(6) total cells cm(-2). Cell morphology was largely dependent on the support material, with spiral bacteria, associated with the infectious form of H. pylori, subsisting in a higher percentage on nonpolymeric substrata. Also, sessile bacteria were generally able to retain the spiral shape for longer when compared with planktonic bacteria, which became coccoid more quickly. The formation of large aggregates, which may act as a protection mechanism against the negative impact of the stressful external environmental conditions, was mostly observed on the surface of copper coupons. However, Syto9 and PI staining indicates that most of H. pylori attached to copper or SS304 have a compromised cell membrane after only 48 h. Cultivability methods were only able to detect the bacteria up to the 2 h exposure-time and at very low levels (up to 500 CFU cm(-2)). CONCLUSIONS: The fact that the pathogen is able to adhere, retain the spiral morphology for longer and form large aggregates when attached to different plumbing materials appeared to point to pipe materials in general, and copper plumbing in particular, as a possible reservoir of virulent H. pylori in water distribution systems. However, the Syto9/PI staining results and cultivability methods indicate that the attached H. pylori cells quickly enter in a nonviable physiological state. SIGNIFICANCE AND IMPACT OF THE STUDY: This represents the first study of H. pylori behaviour in water-exposed abiotic surfaces. It suggests that co-aggregation with the autochthonous heterotrophic consortia present in water is necessary for a longer survival of the pathogen in biofilms associated to drinking water systems.     

Significance of anaerobic preincubation of Helicobacter pylori for measuring metronidazole susceptibility by the Etest.

The Etest is widely used for measuring the susceptibility of Helicobacter pylori to metronidazole. By using 55 H. pylori isolates from 55 patients and a standard H. pylori strain, NCTC11637, we compared metronidazole susceptibility results obtained from the Etest with or without anaerobic preincubation to those obtained from the agar dilution method. Mueller Hinton agar plates supplemented with 5% horse blood were used for both methods. For the Etest, plates were incubated for 72 hr at 35 C under microaerophilic conditions after 0-, 4- or 24-hr periods of anaerobic preincubation. For the agar dilution method, the plates were incubated at the same microaerophilic conditions as those for the Etest. Without anaerobic preincubation for the Etest, 39 of the 56 (70%) H. pylori isolates were categorized as resistant to metronidazole (minimal inhibitory concentration>8 mg/liter), whereas only one of the 56 (1.8%) isolates was resistant according to the agar dilution method. The resistant and susceptible agreement rate was 32%. Four-hour anaerobic preincubation did not alter the readings of the Etest significantly. However, when the Etest was performed with 24-hr anaerobic preincubation, the number of isolates categorized as resistant was reduced to six (11%), improving the agreement rate to 91%. For measuring the metronidazole susceptibility of H. pylori by the Etest, 24-hr anaerobic preincubation is necessary to agree with the results obtained by the agar dilution test.     

Water-induced modulation of Helicobacter pylori virulence properties

While the influence of water in Helicobacter pylori culturability and membrane integrity has been extensively studied, there are little data concerning the effect of this environment on virulence properties. Therefore, we studied the culturability of water-exposed H. pylori and determined whether there was any relation with the bacterium’s ability to adhere, produce functional components of pathogenicity and induce inflammation and alterations in apoptosis in an experimental model of human gastric epithelial cells. H. pylori partially retained the ability to adhere to epithelial cells even after complete loss of culturability. However, the microorganism is no longer effective in eliciting in vitro host cell inflammation and apoptosis, possibly due to the non-functionality of the cag type IV secretion system. These H. pylori-induced host cell responses, which are lost along with culturability, are known to increase epithelial cell turnover and, consequently, could have a deleterious effect on the initial H. pylori colonisation process. The fact that adhesion is maintained by H. pylori to the detriment of other factors involved in later infection stages appears to point to a modulation of the physiology of the pathogen after water exposure and might provide the microorganism with the necessary means to, at least transiently, colonise the human stomach.     

Acidic conditions enhance bactericidal effects of sodium bisulfite on Helicobacter pylori

BACKGROUND: The Brucella broth medium, which is often used for the cultivation of microaerobic bacteria including Helicobacter pylori. It contains sodium bisulfite to decrease oxygen content in the medium. The growth of H. pylori, however, is inhibited by sodium bisulfite. In this study, the effect of sodium bisulfite was compared with several antioxidants and quantified under acidic conditions, mimicking the gastric environment. METHODS: Growth of H. pylori in the presence of several antioxidants was evaluated at OD655 nm. Effect of sodium bisulfite on H. pylori under acidic conditions was evaluated by measuring colony forming units (cfu). RESULTS: Under neutral conditions, sodium bisulfite was a more potent suppressor of H. pylori. Resveratrol, a polyphenol found in wine, exhibited the most potent inhibitory activity. To quantify the effect of sodium bisulfite on H. pylori under acidic conditions, the bacteria were grown at 37 degrees C for 30 minutes in 0.15 mol/l HCl/KCl (pH 2.0) with or without urea and sodium bisulfite. Sodium bisulfite (0.5 mmol/l) did not affect the viability at neutral pH 7.0, however, it killed H. pylori under acidic conditions, even if urea, the key substance enabling H. pylori to survive under acidic conditions, was present. The bacteria, which had been incubated under acidic conditions in the presence of urea, could survive a subsequent 30 minute-incubation at pH 2.0 without urea. Presence of sodium bisulfite, however, in the subsequent 30 minute-incubation, killed the bacteria. CONCLUSIONS: The bactericidal effect of sodium bisulfite on H. pylori was greater under acidic conditions and independent of urease activity.     

Characterization of the respiratory chain of Helicobacter pylori

The respiratory chain of Helicobacter pylori has been investigated. The total insensitivity of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase to antimycin is indicative of the absence of the classical complex I of the electron transfer chain in this bacterium. NADPH-dependent respiration was significantly stronger than NADH-dependent respiration, indicating that this is a major respiratory electron donor in H. pylori. Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase. The activity of succinate-cytochrome c reductase was inhibited by antimycin, implying the presence of a classical pathway from complex II to complex III in this bacterium. The presence of NADH-fumarate reductase (FRD) was demonstrated in H. pylori and fumarate could reduce H2O2 production from NADH, indicating fumarate to be an endogenous substrate for accepting electrons from NADH. The activity of NADH-FRD was inhibited by 2-thenoyltrifluoroacetone. A tentative scheme for the electron transfer pathway in H. pylori is proposed, which may be helpful in clarifying the pathogenesis of H. pylori and in opening new lines for chemotherapy against this bacterium.     

幽门螺杆菌动物模型研究进展

幽门螺杆菌动物模型用于HP相关疾病和HP疫苗作用的研究。常规实验动物包括悉生猪、悉生狗、非人类灵长动物、猫、雪貂、小鼠、大鼠、沙鼠等。猫螺杆菌和雪貂螺杆菌感染也被用于模型研究。最近,转基因小鼠和基因敲除小鼠也被用作幽门螺杆菌动物模型研究。     

Characteristics of Helicobacter pylori natural transformation

For Helicobacter pylori, which exhibits substantial genetic diversity, many strains are naturally competent for transformation by exogenous DNA. To better understand the mechanism of natural transformation and its role in the generation of diversity, we sought to systematically identify factors important for natural transformation in H. pylori. We now show that the highest frequency of H. pylori transformation occurs when DNA is introduced prior to exponential phase growth, and that it is a saturable phenomenon. That transformation can be inhibited by DNA from Helicobacter (H. pylori and Helicobacter bilis) but not Escherichia coli suggests specificity based on DNA source. Finally, the cag island was determined to be unnecessary for high-frequency transformation.     

幽门螺杆菌基因组特征及研究进展

幽门螺杆菌是胃相关疾病：慢性胃炎、消化性溃疡、胃癌和MALT淋巴瘤的一个重要的病原体。其毒力因子包括：尿素酶、鞭毛蛋白、粘附素、细胞毒素相关蛋白和空泡毒素等,通过对全基因序列分析研究,对幽门螺杆菌的致病机制有了进一步的了解。     

Organ-specific autoantibodies in children with Helicobacter pylori infection

BACKGROUND: We compared the prevalence of organ-specific autoantibodies in a group of Helicobacter pylori infected children and a group of uninfected children and investigated the relationship between the presence of relevant autoantibodies and the status of the target organs. PATIENTS AND METHODS: One hundred and twenty-four children with dyspepsia (54 boys, 70 girls; mean age 10.5 years; range 4-19) underwent gastroscopy: 56 had H. pylori infection (31 girls, 25 boys), while 68 (37 girls and 31 boys), were H. pylori-negative. All sera were tested for the presence of: parietal cell autoantibodies (PCA), intrinsic factor autoantibodies (IFA), microsomial autoantibodies, thyroglobulin autoantibodies, islet cell autoantibodies, glutamic acid decarboxylase autoantibodies, adrenal cortex autoantibodies, steroid-producing cell autoantibodies; gastrin, pepsinogen A, pepsinogen C and anti-H. pylori antibodies. The histological features and the ureA and cagA genes were also considered. RESULTS: The frequency of organ-specific autoantibodies was higher in patients with H. pylori infection than in uninfected patients (chi2-test p < .0001). Specifically gastric autoantibodies were significantly higher: seven of the 56 H. pylori-positive children were PCA-positive and one was IFA-positive (chi2-test p = .0004). The presence of autoantibodies was not associated with any clinical or biohumoral signs of disease. CONCLUSIONS: Our study detected a relationship between H. pylori infection in childhood and the presence of organ-specific autoantibodies unassociated with any clinical or biohumoral signs of disease. Helicobacter pylori infection in childhood could trigger the onset of clinical autoimmune gastritis, and/or other clinical autoimmune diseases.