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.     

Oxygen tension regulates reactive oxygen generation and mutation of Helicobacter pylori

Although both bacillary and coccoid forms of Helicobacter pylori reside in human stomach, the pathophysiological significance of the two forms remains obscure. The present work describes the effect of oxygen tension on the transformation and reactive oxygen species (ROS) metabolism of this pathogen. Most H. pylori cultured under an optimum O2 concentration (7%) were the bacillary form, whereas about 80% of cells cultured under aerobic or anaerobic conditions were the coccoid form. The colony-forming unit of H. pylori decreased significantly under both aerobic and anaerobic culture conditions. The bacillary form of H. pylori generated predominantly superoxide radical, whereas the coccoid form generated preferentially hydroxyl radical. Specific activities of cellular respiration, urease, and superoxide dismatase decreased markedly after transformation of the bacillary form to the coccoid form, with concomitant generation of protein carbonyls and 8-hydroxyguanine. The frequency of mutation of cells increased significantly during culture under nonoptimum O2 conditions. These results indicate that ROS generated by H. pylori catalyze the oxidative modification of cellular DNA, thereby enhancing the transformation from the bacillary to the coccoid form. The enhanced generation of mutagenic hydroxyl radicals in the coccoid form might accelerate mutation and increase the genetic diversity of H. pylori.     

Morphological changes and outer membrane protein patterns in Helicobacter pylori during conversion from bacillary to coccoid form

Conversion from bacillary to fully coccoid form via an intermediate U-and V-shaped form has been described in prolonged cultures of H. pylori. This morphological transformation may be the expression of transitory adaptation to a particular environment and may play an important role in antibiotic resistance and the difficulty to eradicate the pathogen. The aim of this study was to evaluate morphological and outer membrane protein changes in H. pylori during ageing-induced conversion to coccoid morphology. We used two H. pylori strains (the reference NCTC 11639 and a fresh clinical isolate) cultivated in microaerophilic environment at 37 degrees C, monitoring their morphological and biochemical evolutions for 11 days. Microscopic examination revealed the passage from spiral to U- and V-shaped form after 5-8 days of incubation, the conversion to coccoid form and the entry into viable but non-culturable state (VBNC) between days 9 and 11. Protein pattern difference appeared at 97.4 to 45 and 30 kDa molecular weight. Biochemical tests demonstrated not only a modification of outer membrane protein profiles, but also an intra-specific variability by comparison between the two analysed strains. Our findings suggest that structural and outer membrane changes associated with coccoid transformation represent a typical response in H. pylori and may constitute a survival strategy in adverse environmental conditions.     

Oxidative cellular damage associated with transformation of Helicobacter pylori from a bacillary to a coccoid form

Exposure to unfavorable conditions results in the transformation of Helicobacter pylori, a gastric pathogen, from a bacillary form to a coccoid form. The mechanism and pathophysiological significance of this transformation remain unclear. The generation of the superoxide radical by H. pylori has previously been shown to inhibit the bactericidal action of nitric oxide, the concentration of which is relatively high in gastric juice. With the use of chemiluminescence probes, both the quality and quantity of reactive oxygen species generated by H. pylori have now been shown to change markedly during the transformation from the bacillary form to the coccoid form. The transformation of H. pylori was associated with oxidative modification of cellular proteins, including urease, an enzyme required for the survival of this bacterium in acidic gastric juice. Although the cellular abundance of urease protein increased during the transformation, the specific activity of the enzyme decreased and it underwent aggregation. Specific activities of both superoxide dismutase and catalase in H. pylori also decreased markedly during the transformation. The transformation of H. pylori was also associated with oxidative modification of DNA, as revealed by the generation of 8-hydroxyguanine, and subsequent DNA fragment. These observations indicate that oxidative stress elicited by endogenously generated reactive oxygen species might play an important role in the transformation of H. pylori from the bacillary form to the coccoid form.     

Characterization of morphological conversion of Helicobacter pylori under anaerobic conditions

Helicobacter pylori (H. pylori), a gram‐negative microaerophilic bacterial pathogen that colonizes the stomachs of more than half of all humans, is linked to chronic gastritis, peptic ulcers and gastric cancer. Spiral‐shaped H. pylori undergo morphologic conversion to a viable but not culturable coccoid form when they transit from the microaerobic stomach into the anaerobic intestinal tract. However, little is known about the morphological and pathogenic characteristics of H. pylori under prolonged anaerobic conditions. In this study, scanning electron microscopy was used to document anaerobiosis‐induced morphological changes of H. pylori, from helical to coccoid to a newly defined fragmented form. Western blot analysis indicated that all three forms express certain pathogenic proteins, including the bacterial cytotoxin‐associated gene A (CagA), components of the cag‐Type IV secretion system (TFSS), the blood group antigen‐binding adhesin BabA, and UreA (an apoenzyme of urease), almost equally. Similar urease activities were also detected in all three forms of H. pylori. However, in contrast to the helical form, bacterial motility and TFSS activity were found to have been abrogated in the anaerobiosis‐induced coccoid and fragmented forms of H. pylori. Notably, it was demonstrated that some of the anaerobiosis‐induced fragmented state cells could be converted to proliferation‐competent helical bacteria in vitro. These results indicate that prolonged exposure to the anaerobic intestine may not eliminate the potential for H. pylori to revert to the helical pathogenic state.

     

The bactericidal and morphological effects of peroxynitrite on Helicobacter pylori

Peroxynitrite (ONOO-) is correlated with the pathogenesis of Helicobacter pylori-induced peptic ulcer diseases. We aimed to investigate the time- and concentration-dependent bactericidal and morphological effects of ONOO- on H. pylori. Authentic ONOO- was synthesized as quenched-flow method. A stock culture of H. pylori NCTC 11637 was exposed to different concentrations of ONOO- (0.1-40 micromol/L) or decomposed ONOO- or fresh medium. Samples were taken at 0, 15, 30, 60, and 120 minutes, for the evaluation of viable bacteria and bacterial morphology with gram strain and transmission electron microscopy. Decomposed ONOO- showed no bactericidal activity against H. pylori. ONOO- application caused a decrease in the number of viable bacteria within the first 15 minutes. The significant conversion of H. pylori from spiral form to coccoid form was determined with 10 micromol/L of ONOO-, and higher concentrations caused lysis of the cells. Separation of cell wall, bleb formation, vacuolization, decrease of secretory granules, and lysis of bacteria were the morphological effects of ONOO- on H. pylori. Because the morphology of the bacteria is one of the important factors in virulence; peroxynitrite-related morphological effects might have an impact in the progress of the H. pylori-induced peptic ulcer diseases.     

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.     

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.     

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.     

Helicobacter pylori– coccoid forms and biofilm formation

Electron microscopic studies have shown that Helicobacter pylori occurs in three stages: spiral forms, coccoid forms and degenerative forms. The spiral forms are viable, culturable, virulent and can colonize experimental animals and induce inflammation. The coccoid forms may also be viable but are nonculturable, less virulent and are less likely to colonize and induce inflammation in experimental animals than the spiral forms. The degenerative forms are pyknotic, nonculturable, coccoid forms of dead H. pylori . These forms cannot be cultured and the cell membrane has disintegrated but gene material can be detected by PCR in water supplies. There is no substantial evidence for viable H. pylori persisting in water supplies. Epidemiological studies suggest that environmental water is a risk factor for H. pylori infection when compared with tap water, and formation of H. pylori biofilm cannot be excluded. Helicobacter pylori does not seem to take part in biofilm formation in the oral cavity even though the bacterium may be detected.     

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.     

Antibacterial activity of highly negative charged polyoxotungstates, K27[KAs4W40O140] and K18[KSb9W21O86], and Keggin-structural polyoxotungstates against Helicobacter pylori

The antibacterial activity of polyoxometalates (PMs) against Helicobacter pylori was investigated based on determinations of minimum inhibitory concentration (MIC) and fractional inhibitory concentration (FIC), time-killing of the bacteria, bacterial morphology and PM-uptake into the bacteria cell. The result of MIC values revealed that, of 13 PMs used in this study, highly negative-charged polyoxotungstates, such as K27[KAs4W40O140] and K18[KSb9W21O86], and Keggin-structural polyoxotungstates exhibited a potent antibacterial activity with the MIC values of less than 256 microg/ml. The former was the most active, and superior to metronidazole (MTZ) against MTZ-susceptible and resistant strains and also to clarithromycin (CLR) against CLR-resistant strains. In contrast, most of polyoxomolybdates showed little antibacterial activity with the MIC values of more than 256 microg/ml. The result of FIC index values indicated that the antibacterial polyoxotungstates had partially synergistic effect in combination with MTZ and CLR but indifferent effect in combination with amoxicillin (AMX). From the results of the time-killing and scanning electron microscope images, K27[KAs4W40O140] and K18[KSb9W21O86] proved the concentration-dependent bactericidal activity with the morphological change from bacillary form to coccoid form, while Keggin-structural K5[SiV(V)W11O40] showed the bacteriostatic activity with small change of morphology to coccoid form. The fluorescent X-ray analysis demonstrated that these polyoxotungstates were taken into the bacteria cell. It is pointed out that the Keggin-structure and/or high negativity polyoxotungstates are an important factor for the antibacterial activity against H. pylori.     

Culture of Helicobacter pylori from stool samples in children

We evaluated two protocols for isolation of Helicobacter pylori in stool from biopsied and nonbiopsied children. Twenty-three child patients whose presumptive positivity or negativity was diagnosed by endoscopy and a rapid urease test at site were used to compare biopsy-based tests with stool-based tests (H. pylori stool antigen test and stool culture). Their gastric activity and bacterial density were graded by the updated Sydney system. Biopsy and stool specimens were cultured on Campy-blood and Belo horizonte agar plates after enrichment in selective Campy-Thio medium. To compare two stool culture protocols, stools from 20 nonbiopsied children were tested by the HpSA test and cultured either as above or after treatment with cholestyramine. Grown colonies were screened by Gram staining, slide agglutination using anti-H. pylori monoclonal IgG; positive isolates were tested by biochemical tests and polymerase chain reaction for H. pylori-specific ureA gene. Coccoid H. pylori was isolated in stool samples from the biopsied patients whose bacterial density was two to four in histology. Their oxidase was slightly positive but became positive after two subcultures, while additional biochemical tests confirmed the isolation of H. pylori. Similar coccoid but oxidase positive H. pylori was isolated from three nonbiopsied children with the protocol of cholestyramine treatment only. The density of bacteria in the stomach may influence the recovery of H. pylori from stool; inactivation of bile with cholestyramine improves the yield in culture and favors isolation of an enhanced metabolic form of bacteria.     

Acute inflammatory response in the stomach of BALB/c mice challenged with coccoidal Helicobacter pylori

An experimental murine model was used to verify the viability and pathogenicity of coccoid Helicobacter pylori. For this purpose, 27 BALB/c mice were inoculated intragastrically with 1 ml broth culture (10(8)organisms/ml) of a coccoid H. pylori clinical isolate. The animals were divided into two groups. Nine were infected on a one-time basis (GA1) and 18 were infected on two consecutive days (GA2). Other 27 mice were inoculated with Brucella broth and divided in the same way; they composed the control group. Mice were killed at 2, 3, 7, 14 and 21 days post inoculation (pi). Fragments of stomach and duodenum were collected, fixed with 12% formalin and stained by hematoxilin-eosin and Giemsa for histopathological examination. Until the 14th()day, only reinfected mice had mild-to-moderate inflammatory infiltrate in the stomach. The infiltration was predominantly lymphomonocytic, although plasma cells and eosinophils could be seen. However, at 21st day, severe eosinophilic infiltration was present in the lamina propria and submucosa of gastric corpus. In subgroup GA1, animals presented lymphomonocytic infiltration in the stomach from 14th()day pi. Our results showed that coccoid H. pylori was able to induce an acute inflammatory response in stomach of reinfected mice since the initial periods of infection.     

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。