幽门螺杆菌球形体回复原形的实验研究

目的对幽门螺杆菌（Helicobacter pylori,Hp）球形体进行体内、外回复原形的比较研究,揭示其潜在的传播途径。方法在布氏肉汤的基础上设计了4种Hp再生培养基,对Hp螺旋体、原生质体及球形体进行体外培养;同时采用30只蒙古沙土鼠（Mongolian gerbil）进行体内感染定植实验,对感染小鼠胃粘膜进行Hp定量培养和组织学检测。结果Hp球形体在4种再生培养基中均未能回复生长;而在感染小鼠的体内却观察到了Hp球形体的回复定植。Hp螺旋体感染组在小鼠胃内的定植密度较高,且胃粘膜下可见大量炎症细胞浸润;而球形体感染组并未见到小鼠胃粘膜组织的明显炎症损伤,且仅有少量回复的螺旋体定植。结论Hp球形体作为一种低水平代谢休眠体,代谢活性及毒力均有所减弱,但仍具有潜在的致病性。本研究支持部分Hp球形体具有活力但体外不能培养成活这一假说,提示＂粪-口＂传播应引起更多的关注。     

Unchanged characteristics of Helicobacter pylori during its morphological conversion.

Helicobacter pylori strains RH 54 and NCTC 11637 were grown in brain-heart infusion broth up to 56 days, and the coccoid form was obtained during prolonged incubation. Two morphological types of coccoids were observed, one of which was electron-dense and had an intact cellular membrane and flagella, indicating that it was likely to be viable. The other coccoid form was sphaeroblast-like and weakly stained, showing features of degeneration. Catalase activity was positive for aged cultures even up to 160 days. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that most of the protein bands appeared to be similar in both the spiral and coccoid forms. In addition, Lewis blood group antigens were detected in cultures of up to 8 weeks. Furthermore, two sets of primers for the vacA and cagA genes were used in polymerase chain reaction, and these two important genes remained conserved in both the spiral and coccoid forms. The present study shows that the coccoid form of H. pylori retained many important characteristics present in the spiral form despite the morphological conversion, and thus supports the notion that some of the coccoid forms of H. pylori are likely to be viable.     

Gastrointestinal colonisation of BALB/cA mice by Helicobacter pylori monitored by heparin magnetic separation

Abstract Immunocompetent and immunodeficient BALB/cA mice were fed orally with 10⁸ colony forming units of 2-day-old spiral or coccoid (12 days old) Helicobacter pylori strain NCTC 11637. Immunocompetent BALB/cA mice were also fed orally with decreasing numbers of spiral or coccoid forms of H. pylori . The gastrointestinal colonisation process was monitored for 34 days post-infection by heparin magnetic separation and subsequent enzyme immunoassay (EIA) for the detection of the H. pylori cells. Both mice types were colonised with H. pylori . The coccoid form of H. pylori gave higher EIA absorbance values and more efficient colonisation in the mice than the spiral form. Immunocompetent BALB/cA mice fed with the coccoid form of H. pylori exhibited an acute inflammation process in histopathological samples from the stomachs. In conclusion, H. pylori can infect both immunocompetent as well as immunodeficient BALB/cA mice and coccoids (viable but non-culturable) obtained after 12 days of culturing can infect BALB/cA mice.     

Alteration in the composition of cholesteryl glucosides and other lipids in Helicobacter pylori undergoing morphological change from spiral to coccoid form

In this analysis of membrane lipid compositions in Helicobacter pylori, the membrane lipid profiles drastically changed during coccoid formation: cholesteryl-6-O-tetradecanoyl-alpha-D-glucopyranoside levels increased, cholesteryl-alpha-D-glucopyranoside and phosphatidyl ethanolamine decreased, and a coordinated increase in cardiolipin and decrease in phosphatidyl glycerol were observed. Cholesteryl-6-O-phosphatidyl-alpha-D-glucopyranoside was hardly detected in the spiral forms in the logarithmic phase, but subsequently increased throughout the coccoid conversion. These results suggest that environmental stresses induce the expression of certain regulatory systems for lipid metabolism in H. pylori, and that the resulting alterations in lipid composition play an important role in inducing the coccoid conversion.     

Oxidative metabolism in nonculturable Helicobacter pylori and Vibrio vulnificus cells studied by substrate-enhanced tetrazolium reduction and digital image processing.

Growing and nonculturable cells of Helicobacter pylori and Vibrio vulnificus were studied for the capacity to reduce tetrazolium salts in order to elucidate the possible physiological basis for the proposed "viable but nonculturable" (VNC) state. Initial difficulties in obtaining consistent reduction of rho-iodonitrotetrazolium violet (INT) by H. pylori led us to develop a method for studying the effect of adding exogenous substrates on these reactions. The established procedure provided a profile of substrate enhancement of oxidative activity revealed by INT reduction which was related to both the identity and physiological state of the organism studied. Representation and interpretation of these enhancement profiles were facilitated by digital image processing. Nonculturable cells of H. pylori produced by carbon and nitrogen starvation in air lost all INT-reducing capacity in 24 h when stored at 37 degrees C, while 99% of those produced at 4 degrees C retained oxidative activity for at least 250 days when tested in the presence but not in the absence of succinate, alpha-ketoglutarate, or aspartate. Activity was detected at similar levels in cells with coccoid and spiral shapes. In contrast, only 1% of nonculturable cells of V. vulnificus, produced under conditions previously reported to induce the VNC state in this organism, retained intrinsic INT-reducing capacity; no substrate-enhanced activity occurred in the remainder of the population. Thus, there was no common pattern of oxidative activity indicative of a VNC state in both test organisms. Nonculturable cells of H. pylori can retain several different oxidative enzyme activities; whether these indicate viability or the persistence of cells as "bags of enzymes" remains to be established.     

球形与螺旋形幽门螺杆菌基因表达差异的研究

目的：从基因转录水平了解球形幽门螺杆菌(Helicobacter pylori,Hp)基因表达的变化。方法：幽门螺杆菌标准株NCTC ll637和2株临床分离株,经抗生素—灭滴灵诱导球变。提取等菌量螺旋形和球形Hp的RNA,RT—PCR扩增Hp25种基因,这些基因包括毒力基因(kat、aphA、rdxA、frxA、cysS、ureB glmM、cagA、vacA、fldA、iceAl、hpaA、fliD、napA、oipA、alpAB、babB、hopZ);看家基因(scoB、atpD、g1nA、recA)和功能不明的外膜蛋白基因(hopW、hopX)。扩增产物经分子定量成像系统进行扫描定量。结果和结论：等菌量的球形HpRNA量比螺旋形Hp减少。NCTC ll637、F44和F49菌株的螺旋菌分别有25、20、19个基因RT—PCR阳性,对应的球形菌相应的基因RT—PCR也为阳性。定量分析结果表明：被检测的基因在球形菌中的表达均比螺旋菌降低。提示球形菌致病性的降低与基因的低表达有关。3株球形菌不同基因表达的变化并不完全一致,表达量变化较小且各菌间较一致的是g1mM、oipA、g1nA;表达量变化较大且菌问较一致的基因是：napA、sodB、recA、fliD。     

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.     

Biofilms in drinking water systems: a possible reservoir for Helicobacter pylori

A laboratory model system was utilised to investigate the persistence of Helicobacter pylori in mixed-species heterotrophic biofilms. A single-stage continuous culture vessel was linked to a modified-Robbins device (mRD) incorporating removable stainless steel coupons. The system was innoculated with H. pylori (NCTC 11637) and the fate of the organism monitored by polymerase chain reaction (PCR) analysis. Helicobacter pylori was detected in biofilm material for a period of up to 192 h. Theoretical washout would have occurred at around 48 h thus detection of H. pylori for a prolonged period after theoretical washout suggested that the organism possessed the ability to persist in the mixed-species heterotrophic biofilm. Preliminary studies using heat-inactivated H. pylori showed that the organism was not detected in biofilm material at any time post-challenge suggesting that the persistence of H. pylori in such material was a phenomenon requiring the organism to be in a viable state. Further investigations to assess the biological basis for the association of H. pylori with drinking water biofilms and the risk that this may pose to public health are being undertaken.     

Non-Helicobacter pylori helicobacters detected in the stomach of humans comprise several naturally occurring Helicobacter species in animals

Besides the well-known gastric pathogen Helicobacter pylori , other Helicobacter species with a spiral morphology have been detected in a minority of human patients who have undergone gastroscopy. The very fastidious nature of these non- Helicobacter pylori helicobacters (NHPH) makes their in vitro isolation difficult. These organisms have been designated ' Helicobacter heilmannii '. However, sequencing of several genes detected in NHPH-infected tissues has shown that the ' H. heilmannii ' group comprises at least five different Helicobacter species, all of them known to colonize the stomach of animals. Recent investigations have indicated that Helicobacter suis is the most prevalent NHPH species in human. This species has only recently been isolated in vitro from porcine stomach mucosa. Other NHPH that colonize the human stomach are Helicobacter felis, Helicobacter bizzozeronii, Helicobacter salomonis and ' Candidatus Helicobacter heilmannii'. In numerous case reports of human gastric NHPH infections, no substantial information is available about the species status of the infecting strain, making it difficult to link the species with certain pathologies. This review aims to clarify the complex nomenclature of NHPH species associated with human gastric disease and their possible animal origin. It is proposed to use the term 'gastric NHPH' to designate gastric spirals that are morphologically different from H. pylori when no identification is available at the species level. Species designations should be reserved for those situations in which the species is defined.     

Coccoid forms of Helicobacter pylori can be viable.

Controversy exists as to whether the coccoid form of Helicobacter pylori can exist in a viable form. Conversion of helical to coccoid morphology occurs in culture over several days. In this study, the morphology was correlated with parameters of genetic integrity in the reference NCTC 11637 strain over 21 days of culture. The capacity to regrow colonies of helical form was demonstrated from a culture where the coccoid form constituted up to 95% and negligible urease activity could be detected. Urease enzyme activity and its mRNA decreased between day 0 and 10 while 26 kD mRNA and 16S rRNA were expressed unchanged for up to 14 and 21 days of culture, respectively. Expression of mRNA for the Cag A gene behaved in a similar fashion to that of urease. No evidence of DNA fragmentation was detected. These data suggest that a viable form of non-urease producing H. pylori exists after short to intermediate culture and that some if not all of these viable bacteria have coccoid morphology.     

Urease Activity and Urea Gene Sequencing of Coccoid Forms of H. pylori Induced by Different Factors

Helicobacter pylori exists in two morphologic forms: spiral shaped and coccoid. The nonculturable coccoid forms were believed to be the morphologic manifestations of cell death for a long time. However, recent studies indicate the viability of such forms. This form of H. pylori is now suspected to play a role in the transmission of the bacteria and is partly responsible for relapse of infection after antimicrobial treatment. Urease activity of H. pylori is an important maintenance factor. Determination of urease activity and possible mutations in the DNA sequences of coccoid bacteria will hence contribute to the understanding of pathogenesis of infections, which these forms might be responsible for. In this study, our aim was to analyze the urease activity and investigate the urease gene sequences of coccoid H. pylori forms induced by different factors with respect to the spiral form. For this purpose, the urease activities of H. pylori NCTC 11637 standard strain and two clinical isolates were examined before and after transformation of the cells to coccoid forms by different methods such as exposure to amoxicillin, aerobiosis, cold starvation, and aging. The effects of these conditions on the urease gene were examined by the amplification of 411-bp ureA gene and 115-bp ureB gene regions by PCR technique and sequencing of the ureA gene. The urease activities of coccoid cells were found to be lower than those of the spiral form. ureA and ureB gene regions were amplified in all coccoid cells by PCR. Inducing the change to coccoid form by different methods was found to have no effect on the nucleotide sequence of the ureA gene. These results show that the urease gene region of coccoid H. pylori is highly protected under various mild environmental conditions.     

Use of autoradiography to assess viability of Helicobacter pylori in water.

Autoradiographic methods have been developed to detect metabolic activity of viable but nonculturable cells of Helicobacter pylori in water. Four strains of H. pylori were studied by using microcosms containing suspensions of 72-h cultures in water. The suspensions of aged, nonculturable cells of H. pylori were incubated with [3H]thymidine for 24 to 72 h, after which the cell suspensions were exposed to Kodak NTB2 emulsion for 3 to 28 days. Each sample was processed with three separate controls to rule out false-positive reactions. The organism remains viable and culturable under these conditions for up to 48 h and, in some cases, 20 to 30 days, depending on physical conditions of the environment. We found that temperature was a significant (P < or equal to 0.01) environmental factor associated with the viability of H. pylori cells in water. Autoradiographs of tritium-labeled cells of H. pylori revealed aggregations of silver grains associated with uptake by H. pylori of radiolabelled substrate. Findings based on the autoradiographic approach give strong evidence supporting the hypothesis that there is a waterborne route of infection for H. pylori. The possibility that H. pylori may persist in water in a metabolically active stage but not actively growing and dividing is intriguing and relevant to public health concerns.     

Detection of Helicobacter pylori by PCR but not culture in water and biofilm samples from drinking water distribution systems in England

AIMS: To investigate treated water distribution systems in England as a source of Helicobacter pylori. METHODS AND RESULTS: Water and biofilms were obtained from 11 domestic and seven educational properties and from hydrants, reservoirs and water meters supplied by three water utilities. Samples were cultured on nonselective and antibiotic containing media combined with immunomagnetic separation concentration. Viable helicobacters were not detected in any of the 151 samples but Helicobacter-specific PCR assays detected DNA in 26% of samples from domestic properties, schools and hydrants with the highest frequency in biofilms (42%). Direct sequencing of six selected amplicons confirmed >95% sequence homology to H. pylori. CONCLUSIONS: While viable helicobacters were not isolated, evidence was obtained for the presence of Helicobacter DNA, including that of H. pylori. Biofilms on surfaces within water distribution systems may act either as sites for the passive accumulation of helicobacters or as potentially important reservoirs of infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings strengthen evidence that H. pylori may be transmitted through drinking water. However, there is currently no evidence that viable cells can survive the disinfection levels used in UK mains supplies and the health risk from this source remains unclear.     

Production of Calcite Nanocrystal by a Urease-Positive Strain of Enterobacter ludwigii and Study of Its Structure by SEM

Helicobacter pylori (H. Pylori) is an actively dividing spiral bacterium that changes to coccoid morphology under stressful environments. The infectivity of the coccoids is still controversial. The aim of this study was to determine the viability and expression of two important virulence genes (babA and cagE), in antibiotic-induced coccoid forms. Three strains of H. pylori, the standard 26695 and two clinical isolates (p1, p2) were converted to coccoid form by amoxicillin. Coccoids were identified according to Gram-staining and microscopic morphology. The viability of the cells was analyzed by flow cytometry. The expression of cagE and babA in coccoid forms were evaluated and compared to the spirals by quantitative PCR assay. The coccoid forms were developed after 72 h exposure of H. pylori to ½ MIC of amoxicillin, and the conversion form was completed (100 %) at 144 h in all of three isolates. Flow cytometry analyses showed that the majority of the induced coccoids (90–99.9 %) were viable. Expression of cagE and babA was seen in coccoids; however, in lower rate (cagE, ~3-fold and babA, ~10-fold) than these in spiral forms. Coccoid forms of two clinical isolates significantly expressed higher rate of cagE and babA than standard 26695 strain (P = 0.01). These results suggest that the induced coccoid form of H. pylori is not a passive entity but can actively infect the human by expression of the virulence genes for long time in stomach and probably play a role in chronic and severe disease.     

Egg Passage of Rodshaped and Coccoid Forms of Helicobacter pylori: Preliminary Studies

Background. We used egg passage of bacteria stored in water to evaluate the culturability of the coccoid form of Helicobacter pylori , as a complement to the results obtained from various animal models. Egg passage was performed, as it is a simple, rapid, and well-characterized old method by which to culture and evaluate culturability of bacteria compared to experiments in animal models. Egg passage has been used in such experiments since 1938 for isolation and growth of, for example, Rickettsiae sp. and Chlamydia sp.
Materials and Methods. The rod-shaped form of H. pylori was produced by plate cultures for 4 and 7 days. The coccoid form of H. pylori was produced by culture on agar plates for 10 days, followed by storage in water. These preparations then were inoculated into the yolk sac of differently aged fertilized eggs.
Results. Positive culture was obtained from 14 of 17 eggs (82%) inoculated with rod-shaped H. pylori compared to 0 of 22 eggs (0%) inoculated with the coccoid form.
Conclusion. Culturability of H. pylori is reduced when it converts into the coccoid form produced by starvation and age followed by storage in water for several weeks at room temperature. Egg passage did not raise the culturability of the coccoid form of H. pylori. Our study demonstrates some clear differences between fresh rods and stored cocci forms of H. pylori in terms of culturability when passed through eggs.     

Characteristics of Helicobacter pylori attachment to human primary antral epithelial cells

Conventional cell lines are commonly used to study infection characteristics of the human gastric pathogen Helicobacter pylori. We sought to investigate bacterial attachment to human antral primary epithelial cells, a cell model that more closely resembles the human stomach than transformed cell lines. Primary cells were infected for 24 and 48 h with H. pylori. Morphological appearance of both the pathogen and the cells as well as features of colonization, attachment and internalization were evaluated by electron microscopy and compared to features observed with cultured AGS cells. H. pylori exhibited various shapes during colonization including the spiral, U-shaped, donut, and coccoid forms. The prevalence of each form seemed to be dependent on the infected donor tissue but, in general, changed with time to the coccoid form. Bacterial cell membranes progressively enlarged and appeared at times to be connected with microvilli. Bacterial attachment occurred to cells that were either unchanged, or had formed cup-like structures. Simultaneously, outer membrane vesicles were increasingly secreted from the bacteria, coinciding with increased cellular damage. We conclude that bacterial shape conversion, adherence and secretion of outer membrane vesicles are features of H. pylori infection. Primary gastric cell cultures closely imitate the antral environment and present an appropriate and useful model to study H. pylori pathogenesis.