Adsorption of dirhamnolipid on four microorganisms and the effect on cell surface hydrophobicity

In this study, adsorption of dirhamnolipid biosurfactant on a Gram-negative Pseudomonas aeruginosa, two Gram-positive Bacillus subtilis, and a yeast, Candida lipolytica, was investigated, and the causality between the adsorption and change of cell surface hydrophobicity was discussed. The adsorption was not only specific to the microorganisms but also depended on the physiological status of the cells. Components of the biosurfactant with different rhamnosyl number or aliphatic chain length also exhibited slight difference in adsorption manner. The adsorption indeed caused the cell surface hydrophobicity to change regularly; however, the changes depended on both the concentrations of rhamnolipid solutions applied and the adsorbent physiological conditions. Orientation of rhamnolipid monomers on cell surface and micelle deposition are supposed to be the basic means of adsorption to change cell hydrophobicity at low and high rhamnolipid concentrations, respectively. This study proposed the possibility to modify cell surface hydrophobicity with biosurfactant of low concentrations, which may be of importance in in situ soil remediation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Supersusceptibility to hydrophobic antimicrobial agents and cell surface hydrophobicity in Branhamella catarrhalis

To clarify the cause of the supersusceptibility of Branhamella catarrhalis to macrolide antibiotics, which are well-known to be inactive to most Gram-negative bacteria, we determined its cell surface hydrophobicity by the partition experiment between water and hydrocarbons. Its cell surface was found to be markedly more hydrophobic than that of Escherichia coli or Pseudomonas aeruginosa cells. This suggested that the outer membrane of B. catarrhalis plays no role as a diffusion barrier towards hydrophobic agents.     

Influence of fluconazole at subinhibitory concentrations on cell surface hydrophobicity and phagocytosis of Candida albicans

Cell surface hydrophobicity (CSH) status influences virulence of Candida albicans and decreases the susceptibility of yeast cells to phagocytic killing. We tested whether subinhibitory concentrations of fluconazole, which is widely used in the treatment and prophylaxis of candidiasis, affect CSH and the susceptibility of C. albicans to enzymatic digestion by glucanase and to phagocytic killing. Treatment of yeast cells with subinhibitory fluconazole concentrations resulted in greater phagocytosis. This effect was independent of CSH but may be related to increased cell wall porosity resulting from alterations in the cell envelope. The use of subinhibitory concentrations of fluconazole in patients with competent phagocytes may contribute to resistance to candidiasis regardless of yeast CSH status.     

Partial characterization of a cell-free hemolytic factor produced by Helicobacter pylori

Abstract Maximum cell-free hemolytic activity of Helicobacter pylori cultured in broth containing 10% horse serum occurred only after the stationary phase of growth was reached, unlike many hemolysins produced by Gram-negative bacteria which are active during exponential growth. This characteristic of the H. pylori hemolytic factor suggested that it might also possess protease activity. However, because no evidence of albumin degradation was found, the hemolysis by cell-free concentrates of H. pylori appears to be due to a unique factor derived from the organism. Because variable hemolysis results were obtained with culture broths lacking albumin or serum, these proteins may act as carriers or stabilizers of the putative hemolysin.     

Suppression of human mononuclear cell response by Helicobacter pylori: Effects on isolated monocytes and lymphocytes

Abstract Helicobacter pylori colonization of the human gastric mucosa causes a long-term, not self-limiting inflammation, suggesting that the microbe has properties to protect itself against the host immune defence system. Recently we were able to demonstrate that H. pylori suppresses the in vitro proliferative response of human peripheral blood mononuclear cells to antigens as well as to mitogens without affecting cell viability. The purpose of this study was to clarify which cell subsets of mononuclear cells are influenced by H. pylori . The use of monocytes which had been pretreated with a soluble cytoplasmic fraction of H. pylori (30 μg ml⁻¹) led to a suppressed proliferation of T cells after PHA-activation. Activation of isolated T cells with PHA and PMA revealed that the proliferative response of lymphocytes could also be inhibited independently of monocytes. The anti-proliferative effect was associated with a reduction of IL-2 receptor (CD25) expression as well as an inhibition of blastogenesis. Furthermore, the spontaneous proliferation of EBV-transformed B cell lines was suppressed in a dose-dependent manner. FACS-analysis of HLA-DR, ICAM-1 and CD14 expression on the surface of monocytes revealed an influence of H. pylori on CD14 expression at a concentration of 30 μg ml⁻¹, while the expression of HLA-DR and ICAM-1 was not affected at this concentration.     

Immunological properties of the cell surface haemagglutinins (sHAs) of Helicobacter pylori strain NCTC 11637

Abstract Rabbits were immunised with stage 1 and stage 2 soluble haemagglutinins (sHA) of Helicobacter pylori strain NCTC 11637 and with rabbit erythrocytes coated with stage 1 sHA. After adsorption of stage 1 sHA on erythrocytes, SDS-PAGE analysis showed that 4 major protein bands were removed from the preparation. The anti-sHA coated erythrocyte serum had the highest HA inhibition titre of 16. Crossed immunoelectrophoresis of the stage 1 sHA, against stage 1 and 2 antisera showed multiple precipitin arcs; however, the anti-sHA coated erythrocyte serum produced only two arcs. One arc produced by the anti-stage 2 serum was absent with the anti-stage 1 serum. This arc could have been produced against a 20 kDa polypeptide which was absent in the stage 1 sHA. The other arc was stronger when compared with that produced by anti-stage 1 serum. These two arcs corresponded to the two arcs produced by the anti-sHA coated erythrocyte serum, which had the highest inhibition titre. The two arcs were markedly reduced in crossed immunoelectrophoresis with an adsorbed stage 1 sHA preparation, which indicates that these arcs were produced against the sHAs.     

幽门螺杆菌ureB基因转染胃上皮细胞及其对细胞的作用

研究幽门螺杆菌 (Helicobacterpylori,Hp)ureB基因重组子转染胃上皮细胞后对胃上皮细胞的作用。用PCR方法从Hp标准株NCTC116 37中获取ureB全长基因 ,将其开放读码框架定向克隆入真核表达载体pcDNA3 1;获得的重组子转染SGC 790 1细胞 ,筛选耐潮霉素的细胞克隆 ,用RT PCR方法检测细胞内ureB基因在转录水平的表达 ;分别用荧光染色技术、MTT、流式细胞术检测UreB对细胞表型、增殖、凋亡及细胞周期的影响。UreB阳性表达的细胞 (SureB)胞膜出芽、细胞皱缩 ;用MTT法检测细胞增殖 ,结果表明 ,SureB细胞与SpcDNA3 1细胞比较 (pcDNA3 1转染的细胞 ) ,生长增殖无显著性差异 (P >0 0 5 ) ,流式细胞术检测细胞凋亡结果显示 ,SureB的凋亡率显著高于SpcDNA3 1(P值为 0 0 0 7) ;细胞周期分析显示 ,SureB细胞有S期比率增高、G2 M、G0 G1 期比率下降的趋势。ureB在培养细胞内的表达可促进细胞凋亡     

幽门螺杆菌相关性胃炎的细胞免疫功能的研究

目的:探讨细胞免疫在Helicobacter pylori(Hp)相关性胃炎的损伤中的作用.方法:检测外周血T淋巴细胞亚群及IL-2、sIL-2R、IL-6、IL-8水平.结果:病例组与对照组比较,CD3 、CD 4和CD 4/CD 8比值及血清IL-2水平均显著降低(P＜0.01),而sIL-2R、IL-6和IL-8水平均显著升高(P＜0.01).结论:细胞免疫在Hp相关性胃炎的损伤中作用明显,检测细胞免疫功能对Hp相关性胃炎的病情监测、疗效观察及预后判定有一定价值.     

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。     

Transmembrane diffusion of hydrophobic antimicrobial agents and cell surface hydrophobicity in Bacteroides fragilis

The transmembrane diffusion of hydrophobic antimicrobial agents, e.g. lincomycin and clindamycin, was examined in Bacteroides fragilis which is sensitive to these agents. The results showed that these agents penetrate efficiently through the outer membrane. Cell surface hydrophobicity measured by the partition assay between water and p-xylene revealed that the cell surface of B. fragilis is more hydrophobic than that of Salmonella typhimurium or Pseudomonas aeruginosa. Furthermore, treatment with low concentrations of surfactant caused cell lysis. These results suggest that the cell surface hydrophobicity in B. fragilis plays an important role in the efficient transmembrane penetration of hydrophobic compounds. This efficiency explains the susceptibility of B. fragilis to hydrophobic antimicrobial agents.     

Mutational analysis of the Helicobacter pylori carbonic anhydrases

In the gastric microenvironment, Helicobacter pylori is exposed to bicarbonate, urea and acid. Here it is demonstrated that both H. pylori carbonic anhydrases (CAs) are required for maintaining urease activity and therefore influence H. pylori urea resistance at neutral pH. Furthermore, the beta-CA is required for acid resistance as indicated by a growth defect of the corresponding mutant at low pH. The alpha- and beta-CA mutants as well as the double mutant were more resistant to bicarbonate, indicating that both enzymes are involved in bicarbonate metabolism. These phenotypes support important CA-functions in H. pylori urea and bicarbonate metabolism and acid resistance. Thus, both CA enzymes might be required for survival in the gastric niche.     

Cell surface characteristics of Helicobacter pylori

Abstract Helicobacter pylori is an important gastroduodenal pathogen of humans. Immunological and structural studies have been performed on the phospholipids, lipopolysaccharides (LPS) and some surface proteins of H. pylori strains. H. pylori LPS has, in general, low immunological activity and this property may aid the survival of this chronic infection. Nevertheless, H. pylori LPS has been found to influence the quality of gastric mucin and to stimulate pepsinogen secretion, thereby contributing to gastric disease. A number of putative adhesins of the bacterium have been described. This multiplicity of adhesins may reflect that H. pylori adherence is a multi-step process involving different interactions, and that different adhesins may mediate adherence to various sites in gastric tissue.     

Medicinal plant extracts variously modulate susceptibility of Escherichia coli to different antibiotics

Antioxidant activity of green and black tea and extracts of medicinal plants and their ability to modulate antibiotic susceptibility in Escherichia coli were studied. Among a number of extracts tested the maximal capacity to scavenge DPPH radicals and chelate iron in chemical tests was found in green and black tea, Arctostaphylos uva-ursi and Vaccinium vitis-idaea. These extracts contained high level of polyphenols and in aerobic conditions exhibited prooxidant features, producing H₂O₂ and inducing expression of the katG gene encoding catalase HPI in E. coli cells. A good correlation between the polyphenol content and the ability of extracts to protect bacteria against peroxide stress was observed (r = 0.88). Polyphenol-rich extracts and iron chelators demonstrated the highest modulating effect on the antibiotic susceptibility by changing the time period before lysis started and by influencing the colony-forming ability of bacteria. The direction of the modulating effect was dependent on nature of antibiotic applied: under treatment with ciprofloxacin and ampicillin the extracts predominantly provided protective effects, while under treatment with kanamycin a bactericidal action was enhanced. Mechanism of modulating action of extracts on bacterial antibiotic susceptibility probably involves antioxidant, preferentially iron-chelating, or prooxidant properties of polyphenols.     

利用杆状病毒表达幽门螺杆菌cagA基因

幽门螺杆菌cagA基因克隆到杆状病毒表达系统的pBlueBacHis2A转移载体中,将重组质粒pBlueBacHis2A-CagA与亲本病毒Bac-N-blue DNA共转染Sf9细胞,以空斑法纯化获得的重组杆状病毒.经PCR法鉴定后进行扩增培养,SDS-PAGE和Western bolt检测结果证实所表达的蛋白为CagA蛋白,间接ELISA分析表明,表达产物可与Hp感染者血清发生特异性的免疫反应.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

Influence of plant polyphenols and medicinal plant extracts on antibiotic susceptibility of Escherichia coli

Aims: To investigate the influence of polyphenols and plant extracts on the susceptibility of Escherichia coli to antibiotics. Methods and Results: Susceptibility of E. coli to antibiotics in the presence of extracts and polyphenols was estimated by the determination of the minimum inhibitory concentrations (MICs). To study gene expression, we used strains of E. coli carrying fusions between promoters of genes katG, sodA, iucC and structural β‐galactosidase gene. Treatment with polyphenols and some plant extracts significantly decreased the antibacterial effects of antibiotics, to a larger extent, ciprofloxacin. The most remarkable protective effect was observed for the extracts of Chamerion (Epilobium) angustifolium, Filipendula vulgaris, Tanacetum vulgare and Serratula coronata. These extracts increased the MICs of ciprofloxacin by four and more times. In case of kanamycin, extracts of Artemisia austriaca and Artemisia pontica increased MICs by four and eight times, respectively. Polyphenol quercetin also caused protective effect against ciprofloxacin, increasing the MIC by four times. A positive correlation was found between protective effects of polyphenols and extracts and their antioxidant activity. Conclusion: Medicinal plant extracts and polyphenols may protect cells of E. coli against antibiotic toxicity. Significance and Impact of the Study: The results of this study may be used to enhance the efficiency of antibacterial therapies.     

Kinetics of substrate oxidation by whole cells and cell membranes of Helicobacter pylori

Abstract Oxygen uptake by Helicobacter pylori cells and membranes was determined. Cells from stirred broth cultures or agar plates, suspended in buffer, possessed a variable and apparently endogenous respiration which could be sustained for several hours. In contrast, oxygen consumption by cells from statically incubated broth cultures, in the absence of added substrate, was transient or undetectable. These latter cells, however, oxidised ethanol, fumarate, glucose, d-lactate, pyruvate and succinate, though glucose-oxidising ability declined rapidly. The K _m s for d-lactate, pyruvate and succinate metabolism were low (≤20 μM) and oxygen uptake was approximately 1.5, 2 and 2 mol per mol substrate respectively, indicating metabolism beyond acetate plus CO₂ and implying the presence of tricarboxylic acid cycle activity. Cell membranes oxidised fumarate, d-lactate, NADH, NADPH and succinate. NADPH oxidation was six times more rapid than that of NADH. Rates of oxygen uptake by cells suspended in buffer with metabolisable substrate were < 20% of those for cells suspended in a brain heart infusion medium. Uninoculated medium consumed significant quantities of oxygen.     

Susceptibility in vitro of Helicobacter pylori to cetylpyridinium chloride

The antimicrobial agent cetylpyridinium chloride (CPC) which is used in therapy of oro-pharyngeal infections and for antiseptic treatment of the oral cavity is active against different bacterial species. Determination of the minimal inhibitory concentration (MIC) using the agar dilution technique revealed that the gastric pathogen Helicobacter pylori in vitro is highly susceptible to CPC as indicated by an MIC of 10 microM (3.4 microg ml(-1)) which was significantly lower than the MIC of CPC against other bacterial species, which were analyzed in comparison to H. pylori. Bacteria of the genus Campylobacter, various Streptococcus spp., Staphylococcus aureus and Escherichia coli showed higher MICs ranging from 100 microM to 2 mM. In summary, this finding renders CPC-containing drugs candidates possibly useful for eradication or for the prevention of transmission of the gastric pathogen.     

Importance of the host genetic background on immune responses to Helicobacter pylori infection and therapeutic vaccine efficacy

In order to investigate the role of host factors in Helicobacter pylori infection and immunity, two different strains of inbred mice, C57BL/6 and BALB/c, were infected with a standard H. pylori strain, SS1. A month later, infected mice were immunized orally with whole-cell lysates of H. pylori SS1 and cholera toxin on days 1, 3, 6, 30, and 54. Ten days after the last immunization, mice were sacrificed and the stomach was collected to assess H. pylori colonization density by quantitative culture. H. pylori SS1 colonization was significantly greater in C57BL/6 than in BALB/c (P<0.02 and P<0.003 at 2 and 13 weeks post-inoculation, respectively). Colonization in C57BL/6 persisted at equivalent levels for 13 weeks but the colonization density in BALB/c decreased significantly during this period. In contrast to the pattern of bacterial colonization, antibody responses following H. pylori SS1 infection were greater in BALB/c than in C57BL/6, suggesting that host factors may modulate the immune responses to H. pylori infection. Following therapeutic immunization, H. pylori colonization in BALB/c mice was also significantly reduced (P<0.03), while no significant differences in bacterial density were observed in C57BL/6. These observations collectively demonstrate the great importance of host factors in H. pylori infection and the development of effective immune responses.     

Application of real-time PCR stool assay for Helicobacter pylori detection and clarithromycin susceptibility testing in Brazilian children

Background: Helicobacter pylori ClariRes assay is a novel commercially available real‐time PCR assay allowing H. pylori detection and clarithromycin susceptibility testing in either gastric biopsy or stool specimens. Objective: The aim of this study was to validate the novel biprobe real‐time assay in stool specimens from 217 dyspeptic children. Methods: DNA from gastric biopsies and stool specimens were obtained and submitted to the biprobe real time assay for H. pylori detection and clarithromycin susceptibility testing. Results: The sensitivity, specificity, and test accuracy were 69, 100 and 93.9% for the detection of H. pylori infection and 83.3, 100 and 95.6%, for detection of clarithromycin resistance. Conclusion: This assay proved to be appropriate for H. pylori clarithromycin susceptibility testing, particularly in children populations where a high prevalence of clarithromycin‐resistant strains is suspected.