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1.
A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the
minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (cat
GC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 × 10−7 and 4.7 × 10−7 transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous
replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly,
both shuttle vectors could also be mobilized efficiently from E. coli into different H.␣pylori recipients, with pHel2 showing an efficiency of 2.0 × 10−5 transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation
of a recA-deficient H. pylori mutant by the cloned H. pylorirecA
+ gene, and the expression of the heterologous green fluorescent protein (GFP) in H.␣pylori demonstrate the general usefulness of␣this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future.
Received: 22 April 1997 / Accepted: 4 November 1997 相似文献
2.
The mode by which Helicobacter pylori, the causative agent of most gastric ulcers, is transmitted remains undetermined. Epidemiological evidence suggests these organisms are waterborne; however, H. pylori has rarely been grown from potential water sources. This may be due to the ability of this organism to rapidly enter the viable but nonculturable (VBNC) state. Our investigation examines the entrance of H. pylori into this state in laboratory cultures and a natural freshwater environment as well as the relationship between morphology and culturability. To this end, membrane diffusion chambers were utilized to expose the cells to the natural fluctuations of a freshwater stream. In both the laboratory and environment, samples were assayed for culturability using plate counts and stained using a LIVE/DEAD BacLight assay for viability and morphological determinations. Additionally, water samples were collected, six environmental parameters were measured, and resuscitation conditions were examined. H. pylori was observed to lose culturability in the laboratory and stream, although viability was maintained. While the results of our study agree with those of previous studies which suggested that there is a transition in morphology from rods to cocci as culturability is lost, the morphological distribution of cells did not change as culturability was lost in the environment. The majority of cells in the VBNC state in the laboratory are cocci; however, all morphological forms were present in the environment. The results of these studies suggest that H. pylori persists in laboratory cultures and the environment in the VBNC state and that cells in this state represent a public health hazard. 相似文献
3.
Nuno M Guimar?es Nuno F Azevedo Maria J Vieira Ceu Figueiredo 《Memórias do Instituto Oswaldo Cruz》2014,109(4):414-419
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. 相似文献
4.
Nutrient Shock and Incubation Atmosphere Influence Recovery of Culturable Helicobacter pylori from Water 下载免费PDF全文
Three different media—Columbia agar, Wilkins-Chalgren agar, and Helicobacter pylori special peptone agar—were prepared in a diluted version and compared to the standard medium formulation in order to study a possible nutrient shock effect observed when recovering H. pylori from water by counting the number of CFU. This same parameter was subsequently used to evaluate the influence of the incubation atmosphere by using a modular atmosphere-controlled system to provide different atmospheres and by employing an established gas generation kit as a control. Both a low nutrient content of the media and a rapidly achieved microaerophilic incubation atmosphere proved to increase the numbers of environment-stressed H. pylori organisms recovered. An atmosphere of 5% CO2, 5% O2, and 3% H2 is recommended, although other atmospheres with a low oxygen concentration are also acceptable. Besides highlighting and assessing the importance of several factors in the culturability of H. pylori, this paper demonstrates the potential ability to develop an optimized technique for recovery of this pathogen from water. 相似文献
5.
Yue-hua Han Wen-zhong Liu Yao-zhou Shi Li-qiong Lu Shu-dong Xiao Qing-hua Zhang 《Journal of microbiology (Seoul, Korea)》2009,47(4):455-465
A Helicobacter pylori whole-genome DNA microarray was constructed to study expression profiles of H. pylori in response to a sudden temperature transfer from 37°C to 20°C. The expression level of the genome at each of four time points
(15, 30, 60, and 120 min) after temperature downshift was compared with that just before cold treatment. Globally, 10.2 %
(n=167) of the total predicted H. pylori genes (n=1636) represented on the microarray were significantly differentially expressed (p<0.05) over a 120 min period after shift to low temperature. The expression profiles of the differentially expressed genes
were grouped, and their expression patterns were validated by quantitative real-time PCR. Up-regulated genes mainly included
genes involved in energy metabolism and substance metabolism, cellular processes, protein fate, ribosomal protein genes, and
hypothetical protein genes, which indicate the compensational responses of H. pylori to temperature downshift. Those genes play important roles in adaption to temperature downshift of H. pylori. Down-regulation of DNA metabolism genes and cell envelope genes and cellular processes genes may reflect damaged functions
under low temperature, which is unfavorable to bacterial infection and propagation. Overall, this time-course study provides
new insights into the primary response of H. pylori to a sudden temperature downshift, which allow the bacteria to survive and adapt to the new host environment. 相似文献
6.
7.
DS Coray JA Heinemann PC Tyrer JI Keenan 《World journal of microbiology & biotechnology》2012,28(5):1871-1880
Helicobacter pylori has high global infection rates and can cause other undesirable clinical manifestations such as duodenal ulcer (DU) and gastric
cancer (GC). Frequencies of re-infection after therapeutic clearance and rates of DU versus GC vary geographically and differ
markedly between developed and developing countries, which suggests additional factors may be involved. The possibility that,
in vivo, lactoferrin (Lf) may play a subtle role in modulating micronutrient availability or bacterial internalisation with
implications for disease etiology is considered. Lf is an iron binding protein produced in mammals that has antimicrobial
and immunomodulatory properties. Some bacteria that regularly colonise mammalian hosts have adapted to living in high Lf environments
and we investigated if this included the gastric pathogen H.
pylori. We found that H.
pylori was able to use iron from fully iron-saturated human Lf (hLf) whereas partially iron-saturated hLf (apo) did not increase
H.
pylori growth. Instead, apo-hLf increased adherence to and internalisation of bacteria into cultured epithelial cells. By increasing
internalisation, we speculate that apo-human lactoferrin may contribute to H.
pylori’s ability to persistence in the human stomach, an observation that potentially has implications for the risk of H.
pylori-associated disease. 相似文献
8.
Edrington TS Callaway TR Hallford DM Chen L Anderson RC Nisbet DJ 《Microbial ecology》2008,55(3):553-560
Fecal prevalence of Escherichia coli O157 in ruminants is highest in the summer decreasing to very low levels in the winter. We hypothesize that this seasonal
variation is a result of physiological responses within the host animal to changing day-length. To determine the effects of
melatonin (MEL) on fecal shedding of E. coli O157:H7 in cattle, eight crossbred beef steers identified as shedding E. coli O157:H7, were allotted to treatment: control or MEL (0.5 mg/kg body weight (BW); 1×) administered orally daily for 7 days.
After a 5-day period of no treatment, a second MEL dose (5.0 mg/kg BW; 10×) was administered daily for 4 days. Fecal samples
were collected daily for qualification of E. coli O157:H7. No differences (P > 0.10) were observed in the percentage of E. coli O157:H7 positive fecal samples in steers receiving the 1× MEL dose, however the 10× dose decreased (P = 0.05) the percentage of fecal samples E. coli O157:H7 positive. Serum MEL concentrations were higher in the 1×, but not 10×, treated animals compared to control animals.
Although it is difficult to explain, this may be a result of decreasing day-length increasing serum melatonin concentrations
that may have masked any treatment effect on serum melatonin. In a second similar experiment, a second group of cattle (heifers
and steers) were administered tryptophan (TRP) over a 17-day experimental period (5 g/head/day for 10 days followed by 10 g/head/day
for 7 days). Tryptophan had no effect (P > 0.20) on the percentage of fecal samples positive for E. coli O157. Serum TRP (P < 0.05), but not MEL (P > 0.20), concentrations were elevated in TRP-treated animals. The decrease in the number of positive fecal samples observed
in the first experiment, may be related to gastrointestinal MEL, affected by the 10×, but not 1× MEL dose. 相似文献
9.
Li N Xu X Xiao B Zhu ED Li BS Liu Z Tang B Zou QM Liang HP Mao XH 《Molecular biology reports》2012,39(4):4655-4661
MicroRNAs have been implicated as a central regulator of the immune system. We have previously reported that Helicobacter pylori (H. pylori) was able to increase the expression of miR-146a, and miR-146a may negatively regulate H. pylori-induced inflammation, but the exact mechanism of how H. pylori contribute the induction of miR-146a is not clear. Here, we attempted to assess the role of H. pylori related proinflammatory cytokines including interleukin (IL)-8, tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, and
cytotoxin-associated gene A (CagA) virulence factor on the induction of miR-146a. We found that IL-8, TNF-α, and IL-1β could
contribute to the induction of miR-146a in gastric epithelial cell HGC-27 in NF-κB-dependent manner, while the induction of
miR-146a upon H. pylori stimulation was independent of above proinflammatory cytokines. Furthermore, overexpression of miR-146a reduced H. pylori—induced IL-8, TNF-α, and IL-1β. However, CagA had no effect on the miR-146a induction. Taken together, our study suggest that proinflammatory cytokines IL-8,
TNF-α, and IL-1β could contribute to the induction of miR-146a during H. pylori infection, while CagA is not necessarily required for miR-146a induction. miR-146a may function as novel negative regulators
to modulate the inflammation. 相似文献
10.
Helicobacter pylori (H pylori) is the main risk factor for gastric cancer (GC). In recent years, many studies have addressed the effects of H pylori itself and of H pylori‐induced chronic inflammation on DNA damage. Unrepaired or inappropriately repaired DNA damage is one possible carcinogenic mechanism. We may conclude that H pylori‐induced DNA damage is one of the carcinogenic mechanisms of GC. In this review, we summarize the interactions between H pylori and DNA damage and the effects of H pylori‐induced DNA damage on GC. Then, focusing on oxidative stress, we introduce the application of antioxidants in GC. At the end of this review, we discuss the outlook for further research on H pylori‐induced DNA damage. 相似文献
11.
Helicobacter pylori is a highly successful human-specific gastric pathogen that infects up to 50% of the world’s population. Virulent H. pylori isolates harbor the cytotoxin-associated genes pathogenicity island (cag-PAI), which encodes a type IV secretion system that translocates bacterial effector (e.g., CagA oncoprotein) molecules into
host cells. Although some cag-PAI genes are shown to be required for CagA delivery or localization, the majority have no known function. In the current
study, the authors performed a cell components fractionation assay and showed that CagI, one of the cag-PAI proteins located in the bacterial membrane, was not translocated into host cells. The homologous recombination method
then was used to construct the isogenic mutant of H. pylori cagI, and the translocation assay was performed. The results showed that the isogenic mutant of H. pylori NCTC 11637 cagI could cause a reduction in the degree of CagA translocation. Overall, the results suggested that CagI might be an accessory
component of the CagA secretion system not translocated into host cells and that it is located in the bacterial membrane. 相似文献
12.
The hypothesis that the Ajime-loach, Niwaella delicata, is guided to groundwater seepages by a positive thermotaxis in autumn, was tested by a field investigation and aquarium-based
experiments. A total of 763 individuals of N. delicata were captured from October to November in a groundwater trap in the Yasu River, Shiga Prefecture. Niwaella delicata began to be captured as the temperature of the surface water fell to 15.8° ± 1.1°C (mean ± SD) and that of the groundwater
to 15.5° ± 0.9°C. Groundwater was often warmer than surface water at night or occasionally all day, and the difference in
temperature reached a maximum of 1.3°C at the night on 5 November. For the diel pattern of captures, nocturnal capture was
higher than diurnal capture when the groundwater was warmer at night and colder during the daytime, whereas both diurnal and
nocturnal captures were high when the groundwater was always warmer than the surface water. The aquarium-based experiments
showed that N. delicata choose warmer water, ranging from 18.4° to 22.2°C, just before the capture period in the Yasu River, and are sensitive to
differences in water temperature of 1.3° ± 0.1°C. Although the present results broadly support the hypothesis, a part of the
results indicates that water temperature gradients may not be the only factor involved in the groundwater selection of N. delicata. 相似文献
13.
Background. Recent study has demonstrated that β‐lactamase inhibitors including clavulanate, sulbactam and tazobactam have an vitro antibacterial effect on Helicobacter pylori. Here we describe the relationship between viability and cell profiles of H. pylori exposed to β‐lactamase inhibitors and some antibiotics in a short‐time course. Materials and methods. The antibacterial effects of β‐lactamase inhibitors including clavulanate, sulbactam and tazobactam on the bacterial viability of and morphological changes in H. pylori ATCC43504 were examined. Results. The β‐lactamase inhibitors such as clavulanate and sulbactam alone decreased the viable counts of H. pylori, depending on the antibiotic concentrations. Exposure to these β‐lactamase inhibitors resulted in morphological changes of cell shape, cell‐wall disintegration and cell lysis. Among these β‐lactamase inhibitors, clavulanate was the most active, causing a decrease in viable counts and morphological changes such as short filamentous to sphaeroplast formation and lysis. One × minimum inhibitory concentration (MIC) of amoxicillin plus 1 × MIC of clavulanate decreased viable counts effectively compared with 1 × MIC of amoxicillin or 1 × MIC of clavulanate alone, and induced morphological changes of cell shape and cell wall. Conclusion. Our results suggest that the β‐lactamase inhibitors alone have concentration‐dependent antibacterial activities against H. pylori and affect the morphology of the cell shape and the cell wall in vitro. 相似文献
14.
Beneduce L. Tarantino D. Spano G. Libergoli M. Labonia Maria Massa S. 《World journal of microbiology & biotechnology》2003,19(5):505-508
The mortality of a clinical Helicobacter pylori strain was assessed by inoculating it in untreated well water, filtered well water, and autoclaved well water. Two different temperatures (5 and 25 °C) were used during the experimental period. Because Escherichia coli is commonly used as indicator of faecal pollution of water, we compared the survival of H. pylori using E. coli as indicator of its persistence. H. pylori was not culturable 48 h after inoculation, whereas the population of E. coli, monitored at the same temperature, decreased slowly, especially in filtered water. In untreated water, both H. pylori and E. coli survived less well than in filtered and autoclaved water. In general the survival of H. pylori and E. coli was better in filtered water than in autoclaved water and the ability of H. pylori to survive several days in water at 5 °C is reported, supporting the observation that H. pylori survives better at 5 °C than at higher temperature. This suggests a possible faecal–oral transmission of H. pylori in the presence of a contaminated water. 相似文献
15.
M. J. Tipton F. S. C. Golden C. Higenbottam I. B. Mekjavic C. M. Eglin 《European journal of applied physiology and occupational physiology》1998,78(3):253-257
The initial responses to cold-water immersion, evoked by stimulation of peripheral cold receptors, include tachycardia, a
reflex inspiratory gasp and uncontrollable hyperventilation. When immersed naked, the maximum responses are initiated in water
at 10°C, with smaller responses being observed following immersion in water at 15°C. Habituation of the initial responses
can be achieved following repeated immersions, but the specificity of this response with regard to water temperature is not
known. Thirteen healthy male volunteers were divided into a control (C) group (n = 5) and a habituation (H) group (n = 8). Each subject undertook two 3-min head-out immersions in water at 10°C wearing swimming trunks. These immersions took
place at a corresponding time of day with 4 days separating the two immersions. In the intervening period the C group were
not exposed to cold water, while the H group undertook another six, 3-min, head-out immersions in water at 15°C. Respiratory
rate (f
R), inspiratory minute volume (V˙
I) and heart rate (f
H) were measured continuously throughout each immersion. Following repeated immersions in water at 15°C, the f
R, V˙
I and f
H responses of the H group over the first 30 s of immersion were reduced (P < 0.01) from 33.3 breaths · min−1, 50.5 l · min−1 and 114 beats · min−1 respectively, to 19.8 breaths · min−1, 26.4 l · min−1 and 98 beats · min−1, respectively. In water at 10°C these responses were reduced (P < 0.01) from 47.3 breaths · min−1, 67.6 l · min−1 and 128 beats · min−1 to 24.0 breaths · min−1, 29.5 l · min−1 and 109 beats · min−1, respectively over a corresponding period of immersion. Similar reductions were observed during the last 2.5 min of immersions.
The initial responses of the C group were unchanged. It is concluded that habituation of the cold shock response can be achieved
by immersion in warmer water than that for which protection is required. This suggests that repeated submaximal stimulation
of the cutaneous cold receptors is sufficient to attenuate the responses to more maximal stimulation.
Accepted: 6 February 1998 相似文献
16.
Heavy water (H218O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been
reported for the 18O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% 18O for DNA extracted from soil incubated with the addition of H218O. Four experiments were conducted to test how the atom% 18O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1)
time, (2) nutrients, (3) soil moisture, and (4) atom% 18O of added H2O. In the time series experiment, the atom% 18O of DNA increased linearly (R
2 = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R
2 = 0.991, p = 0.006) between the log10 of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log10 of the atom% 18O of DNA. For the experiment where soil moisture was manipulated, the atom% 18O of DNA increased with higher soil moisture until soil moisture reached 30%, above which 18O enrichment of DNA declined as soils became more saturated. When the atom% 18O for H2O added was varied, there was a positive linear relationship between the atom% 18O of the added water and the atom% 18O of the DNA. Results indicate that quantification of 18O incorporated into DNA from H218O has potential to be used as a proxy for microbial growth in soil. 相似文献
17.
Israt Farhana Zenat Zebin Hossain Suhella Mohan Tulsiani Peter Kjær Mackie Jensen Anowara Begum 《World journal of microbiology & biotechnology》2016,32(9):146
It is well established that the contamination sources of cholera causing bacteria, Vibrio cholerae, are water and food, but little is known about the transmission role of the fomites (surfaces that can carry pathogens) commonly used in households. In the absence of appropriate nutrients or growth conditions on fomites, bacteria have been known to assume a viable but non-culturable (VBNC) state after a given period of time. To investigate whether and when V. cholerae O1 assumes such a state, this study investigated the survival and viable quantification on a range of fomites such as paper, wood, glass, plastic, cloth and several types of metals under laboratory conditions. The fomites were inoculated with an outbreak strain of V. cholerae and its culturability was examined by drop plate count method at 30 min intervals for up to 6 h. For molecular detection, the viable/dead stain ethidium monoazide (EMA) which inhibits amplification of DNA from dead cells was used in combination with real-time polymerase chain reaction (EMA-qPCR) for direct quantitative analyses of viable V. cholerae at 2, 4, 6, 24 h and 7 day time intervals. Results showed that V. cholerae on glass and aluminum surfaces lost culturability within one hour after inoculation but remained culturable on cloth and wood for up to four hours. VBNC V. cholerae on dry fomite surfaces was detected and quantified by EMA-qPCR even 7 days after inoculation. In conclusion, the prolonged survival of V. cholerae on various household fomites may play vital role in cholera transmission and needs to be further investigated. 相似文献
18.
Characterization of morphological conversion of Helicobacter pylori under anaerobic conditions 下载免费PDF全文
Sayaka Hirukawa Hiroshi Sagara Satoshi Kaneto Tomoyo Kondo Kotaro Kiga Takahito Sanada Hiroshi Kiyono Hitomi Mimuro 《Microbiology and immunology》2018,62(4):221-228
19.
Kottakis F Lamari F Matragkou Ch Zachariadis G Karamanos N Choli-Papadopoulou T 《Amino acids》2008,34(3):413-420
Summary. Arabino-Galactan Proteins (AGPs) were isolated from Chios mastic gum (CMG) by using a buffer containing 0.1 M NaCl, 20 mM
Tris–HCl, pH 7.5. Protein analytical methods, combined with specific procedures for carbohydrate characterization, indicated
the presence of highly glycosylated protein backbone. In particular, staining by Yariv reagent of the electrophoretically
separated molecules revealed the existence of arabinose and galactose and such a modification is characteristic for AGPs.
After experiments involving extensive dialysis of the isolated extracts against water and atomic absorption, there was evidence
of the existence of zinc ions that are probably covalently bound to the AGPs. By using anion-exchange chromatography, capillary
electrophoresis, colorimetric methods and GC-MS, it was found that the extracts were separated into three major populations
(A, B, and C), which were consistent with their respective negative charge content namely, uronic acid. The characterization
of neutral sugars that was investigated with GC-MS showed the existence of arabinose and galactose in different amounts for
each group.
Experiments concerning the inhibition of growth of Helicobacter pylori in the presence of AGPs, as is shown for other CMG constituents, showed that the extracts of at least 1.4 g CMG affected
the viability of the bacterium. There is no evidence as to whether the AGPs provoke abnormal morphologies of H. pylori, as is reported for the total CMG, or for O-glycans that possess terminal α1, 4-linked N-acetylglucosamine and are expressed
in the human gastric mucosa; this has to be further investigated.
Authors’ address: Theodora Choli-Papadopoulou, Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki,
TK 54124 Thessaloniki, Greece 相似文献
20.
Helicobacter pylori is a Gram-negative pathogen that colonizes the gastric epithelium of 50–60% of the world’s population. Approximately one-fifth of the infected individuals manifest severe diseases such as peptic ulcers or gastric cancer. H. pylori infection has proven difficult to cure despite intensive antibiotic treatment. One possible reason for the relatively high resistance to antimicrobial therapy is the ability of H. pylori to reside inside host cells. Although considered by most as an extracellular pathogen, H. pylori can invade both gastric epithelial cells and immunocytes to some extent. The intracellular survival of H. pylori has been implicated in its ability to persist in the stomach, evade host immune responses and resist eradication by membrane-impermeable antibiotics. Interestingly, recent evidence suggests that macroautophagy, a cellular self-degradation process characterized by the formation of double-membraned autophagosomes, plays an important role in determining the intracellular fate of H. pylori. Detailed understanding of the interaction between H. pylori and host cell autophagic processes is anticipated to provide novel insights into the molecular mechanisms of macroautophagy and H. pylori pathogenesis, opening new avenues for the therapeutic intervention of autophagy-related and H. pylori-related disorders. 相似文献