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.     

Inactivation of the Bactericidal Activity of Human Serum by Liquoid (Sodium Polyanetholsulfonate

Four serum-sensitive strains of Escherichia coli were exposed to 10, 20, and 50% fresh, heat-inactivated, and fresh human serum to which had been added Liquoid at a final concentration of 0.05, 0.025, 0.0125 and 0.006%. It was found that 50% fresh serum (in nutrient, Mueller-Hinton, thioglycolate, or Trypticase Soy Broth) killed more than 10(4) organisms/ml within 3 min, whereas 20 and 10% fresh serum required up to 20 and 40 min, respectively, to kill a comparable number of organisms. To neutralize the activity of 50% fresh serum, 0.0125% Liquoid had to be added, whereas an 0.006% final concentration of Liquoid was sufficient to antagonize the activity of 10 and 20% serum. However, when exposing extremely small bacterial inocula to fresh serum, at least 0.025% Liquoid was necessary to abolish the serum-bactericidal activity of 20 and 50% fresh serum. Liquoid had to be added to 50% fresh serum within seconds to prevent the killing of the majority of test organisms derived from small inocula. It is recommended that blood samples drawn from septicemic or bacteremic patients be aseptically added to a suitable broth which contains at least 0.025% Liquoid in order to improve the chances of isolating pathogens present in small numbers.     

The Stability of Complement-Mediated Bactericidal Activity in Human Serum against Salmonella

The complement cascade includes heat-labile proteins and care is required when handling serum in order to preserve its functional integrity. We have previously used a whole human serum bactericidal assay to show that antibody and an intact complement system are required in blood for killing of invasive isolates of Salmonella. The aim of the present study was to evaluate the conditions under which human serum can be stored and manipulated while maintaining complement integrity. Serum bactericidal activity against Salmonella was maintained for a minimum of 35 days when stored at 4°C, eight days at 22°C and 54 hours at 37°C. Up to three freeze-thaw cycles had no effect on the persistence of bactericidal activity and hemolytic complement assays confirmed no effect on complement function. Delay in the separation of serum for up to four days from clotted blood stored at 22°C did not affect bactericidal activity. Dilution of serum resulted in an increased rate of loss of bactericidal activity and so serum should be stored undiluted. These findings indicate that the current guidelines concerning manipulation and storage of human serum to preserve complement integrity and function leave a large margin for safety with regards to bactericidal activity against Salmonella. The study provides a scheme for determining the requirements for serum handling in relation to functional activity of complement in other systems.     

Rapid Method for Determining Serum Bactericidal Activity

To screen large numbers of sera, a method was devised which utilizes the Steers-Foltz replicator which is usually used to determine minimal inhibitory concentration for antibiotics. Each of the wells (9 by 15 mm) of the replicator is filled with 0.06 ml of serum, 0.02 ml of a 10(5) suspension of organisms, and 0.02 ml of diluent (tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 8.4). The mixtures are incubated for 3 h, and samples are taken at 0, 1, 2, and 3 h by stamping duplicate nutrient agar plates (approximately 0.04 ml from each well). Plates are incubated overnight, and bactericidal activity is estimated by visual inspection of bacterial growth at each site for each sampling time. Results obtained with 28 serum-organism pairs paralleled standard pipetting-pour plate methods. The replicator method for determining bactericidal activity allows for the testing of a large number of samples and requires negligible amounts of serum.     

Resistance of Pseudomonas pseudomallei to Normal Human Serum Bactericidal Action

The effect of human normal serum (HNS) on Pseudomonas pseudomallei was determined. It is apparent from our data that the organism is resistant to the normal serum bactericidal mechanism. Ancillary experiments to confirm this serum-resistant property of P. pseudomallei were done by examining the effects of growth phase conditions of the bacteria (i.e., logarithmic and stationary phases) and different buffered systems used as diluent in our bactericidal assay. Results obtained showed similar degree of resistance to serum bactericidal killing by 5 strains of the organisms tested. The possible survival advantage of serum-resistant property to P. pseudomallei as bacterial pathogens known to invade the blood stream is discussed.     

Serum Bactericidal Activity of Trovafloxacin Against Selected Anaerobic Pathogens

Trovafloxacin is a fluoroquinolone antimicrobial with improved in vitro activity against many anaerobic bacteria. We investigated the serum bactericidal activity of trovafloxacin against isolates of Bacteroides fragilis, Bacteroides thetaiotaomicron, Clostridium perfringens, and Peptostreptococcus magnus using a broth microdilution method. All procedures were performed in an anaerobic chamber. A single 200 mg oral dose of trovafloxacin was administered to six healthy volunteers and serum samples were obtained at 0, 2, 4, 6, 8, 10, 12 and 24 h post-dose. Bactericidal activity of these samples demonstrated that at 2 h all samples showed bactericidal activity against all four isolates. Prolonged bactericidal activity (12 to 24 h) was observed against three of the four isolates. Bactericidal activity was not observed after the first sampling period for the B. thetaiotaomicron strain. In this pharmacodynamic model, we found that trovafloxacin provided serum bactericidal activity against several common anaerobic pathogens associated with clinical infections.     

Adherence of Helicobacter pylori to Abiotic Surfaces Is Influenced by Serum

Helicobacter pylori bacteria cultured in a chemically defined medium without serum readily adhere to a variety of abiotic surfaces. Growth produces microcolonies that spread to cover the entire surface, along with a planktonic subpopulation. Serum inhibits adherence. Initial attachment is protein mediated, but other molecules are responsible for more permanent attachment.     

Invasiveness of Helicobacter pylori into Human Gastric Mucosa

Background. Helicobacter pylori has generally been observed only in the gastric mucous layer or in the spaces between gastric mucus -s ecreting cells and not in the gastric epithelial cells or in the lamina propria. The purpose of this study is to determine whether H. pylori invades the gastric mucosa, using an immunoelectron microscopical examination of human gastric mucosa infected with H. pylori.
Materials and Methods. Five hundred gastric antral biopsy specimens were fixed in a periodate-lysin-paraformaldehyde solution, embedded in Lowicryl, sectioned, and examined with a light microscope. One hundred specimens moderately or severely infected with H. pylori were selected and were incubated with polyclonal rabbit anti– H. pylori antibody. The specimens were washed, incubated with 20 nm of colloidal gold–conjugated goat anti–rabbit IgG, stained with uranyl acetate and lead citrate, and observed with a transmission electron microscope.
Results. In one case, a bacterium was observed within the cytoplasm of a gastric mucus -s ecreting cell; in another case, a few bacteria were observed within the cytoplasm of a stromal cell in the lamina propria. The bacteria could be differentiated from degenerated intracellular organelles by gold particles attached to the bacteria.
Conclusion. H. pylori rarely invade the lamina propria and gastric cells.     

Combined Activity of Azithromycin and Lansoprazole Against Helicobacter pylori

Background. Due to its unique pharmacokinetic properties, azithromycin may be an attractive combination partner for H. pylori eradication regimens. However, up to 15% of clinical isolates are primarily resistant to azithromycin as well as to other macrolide antibiotics. Combination therapy with lansoprazole, a proton pump inhibitor known to have intrinsic antibacterial activity against H. pylori , may be useful to counteract such resistance. We therefore evaluated the combined effects of azithromycin and lansoprazole in vitro.
Materials and Methods. Minimal inhibitory concentrations (MICs) of azithromycin and lansoprazole alone and in combination were determined for 106 clinical H. pylori isolates by means of an agar dilution technique. Killing kinetics of seven isolates were also studied in fluid medium.
Results. MIC values for 50 and 90% of the isolates (MIC₅₀, MIC₉₀) were 0.19 and 0.5 mg/l for azithromycin, and 44.5 and 104 mg/l for lansoprazole. Nine strains (8.5%) had an MIC of azithromycin ≥ 16 mg/l and were regarded as resistant. An additive interaction between the two drugs was found in 72 (68%), and indifferent effects in 24 strains (23%). Three of 9 azithromycin-resistant strains regained sensitivity in the presence of lansoprazole. In fluid culture, synergism between the two drugs occurred in 6 out of 7 strains tested.
Conclusion. In the majority of strains, lansoprazole and azithromycin interacted in an additive or synergistic manner depending on the test method employed. Addition of lansoprazole restored in vitro sensitivity to azithromycin in 3 out of 9 azithromycin-resistant strains. Such effects may enhance the elimination of H. pylori during clinical eradication therapy.     

Susceptibility to Helicobacter pylori infection: results of an epidemiological investigation among gastric cancer patients

The aim of this study was to identify the clinical, demographic, lifestyle factors and selected genetic polymorphisms that affect the susceptibility towards Helicobacter pylori (H. pylori) infection in gastric cancer patients. Histological confirmed gastric adenocarcinoma cases that underwent curative gastrectomy between 2002 and 2012 were included. Gastric biopsy samples were obtained to determine the H. pylori status, and further cagA status and vacA m and s genotypes by polymerase chain reaction. Patients were interviewed with structured questionnaires, and blood samples were collected for EPHX1, GSTM1, GSTT1, IL1B, IL1-RN, MTHFR and p53 genotyping. Proportions were compared in univariate analysis, while the relation between putative risk factors and H. pylori status and genotype were measured using logistic regression analysis. One hundred forty-nine gastric cancer patients were included, of which 78.5 % were H. pylori positive. Among positive patients 50 % were cagA+, 72.5 % vacA m1 and 80.7 % vacA s1. The presence of cagA was less frequent among vacA m1 (p = 0.031) and vacA s1 (p = 0.052) subtypes. The presence of father history for any cancer was a significant risk factor for H. pylori infection [adjusted odds ratio (OR) = 8.18, 95 % confidence interval (CI) 1.04–64.55]. EPHX1 exon 3 T > C (OR = 0.35, CI 95 % 0.13–0.94), IL1B-511 T > C (OR = 0.38, CI 95 % 0.15–0.97) and IL1-RN VNTR (OR = 0.19, CI 95 % 0.06–0.58) polymorphisms were protective towards H. pylori infection in the univariate analysis. Wine consumption was associated with higher risk of carrying the H. pylori vacA m1 virulent subtype (p = 0.034). Lastly, cardiovascular diseases were less common among cagA positive subjects (p = 0.023). Father history of any cancer is a risk factor for H. pylori infection. Polymorphisms in IL1B-511, IL1-RN and EPHX1 exon 3 genes might be protective towards H. pylori infection.     

Contraselectable Streptomycin Susceptibility Determinant for Genetic Manipulation and Analysis of Helicobacter pylori

Many Helicobacter pylori genetic studies would benefit from an ability to move DNA sequences easily between strains by transformation and homologous recombination, without needing to leave a conventional drug resistance determinant at the targeted locus. Presented here is a two-gene cassette that can be selected both (i) against, due to a Campylobacter jejuni rpsL gene (dominant streptomycin susceptibility in cells also carrying an rpsL-str^r allele), and (ii) for, due to an erm gene (erythromycin resistance). This rpsL,erm cassette's utility was assessed by using it to replace four gene loci (mdaB, frxA, fur, and nikR) in four streptomycin-resistant [Str^r] strain backgrounds (derivatives of 26695, SS1, X47, and G27MA). The resultant 16 strains (phenotypically erythromycin resistant [Erm^r] and Str^s) were each transformed with wild-type genomic DNAs, and Str^r derivatives were selected. The desired Erm^s Str^r isolates were obtained at frequencies that ranged from 17 to 96% among Str^r transformants, with the Erm^s yield apparently depending on the strain background and genome location of the targeted locus. The ease of isolating unmarked transformants described here should be valuable for many H. pylori molecular genetic and evolutionary analyses.     

Role of Immune Serum in the Killing of Helicobacter pylori by Macrophages

Background: Helicobacter pylori infection can lead to the development of gastritis, peptic ulcers and gastric cancer, which makes this bacterium an important concern for human health. Despite evoking a strong immune response in the host, H. pylori persists, requiring complex antibiotic therapy for eradication. Here we have studied the impact of a patient’s immune serum on H. pylori in relation to macrophage uptake, phagosome maturation, and bacterial killing. Materials and Methods: Primary human macrophages were infected in vitro with both immune serum‐treated and control H. pylori. The ability of primary human macrophages to kill H. pylori was characterized at various time points after infection. H. pylori phagosome maturation was analyzed by confocal immune fluorescence microscopy using markers specific for H. pylori, early endosomes (EEA1), late endosomes (CD63) and lysosomes (LAMP‐1). Results: Immune serum enhanced H. pylori uptake into macrophages when compared to control bacteria. However, a sufficient inoculum remained for recovery of viable H. pylori from macrophages, at 8 hours after infection, for both the serum‐treated and control groups. Both serum‐treated and control H. pylori phagosomes acquired EEA1 (15 minutes), CD63 and LAMP‐1 (30 minutes). These markers were then retained for the rest of an 8 hour time course. Conclusions: While immune sera appeared to have a slight positive effect on bacterial uptake, both serum‐treated and control H. pylori were not eliminated by macrophages. Furthermore, the same disruptions to phagosome maturation were observed for both serum‐treated and control H. pylori. We conclude that to eliminate H. pylori, a strategy is required to restore the normal process of phagosome maturation and enable effective macrophage killing of H. pylori, following a host immune response.     

Bactericidal effect of bovine normal and immune serum, colostrum and milk against Helicobacter pylori

Serum and colostrum but not post-colostral milk from non-immunized Friesian cows was found highly bactericidal for Helicobacter pylori NCTC 11637. This bactericidal activity was destroyed by heating at 56°C for 30 min and restroed by the addition of fetal calf serum as a source of complement, indicating that the bactericidal effect was probably dependent on an antibody-complement system. Systemic, serial immunization of non-lactating, pregnant cows with H. pylori resulted in high specific antibody titres in serum and colostrum. No titres were found in post-colostral milk, even after booster-immunization during lactation. Immunization did not enhance the bactericidal activity of serum and colostrum, but increased it in post-colostral milk. The bactericidal activity was not correlated with titres of specific antibody or with IgG concentrations.     

Human polymorphonuclear leukocytes are sensitive in vitro to Helicobacter pylori vaca toxin

BACKGROUND: Interactions between bacterial components and polymorphonuclear leukocytes (PMNL) play a major pathogenic role in Helicobacter pylori-associated diseases. Activation of PMNL can be induced by contact with whole bacteria or by different H. pylori products released in the extracellular space either by active secretion or by bacterial autolysis. Among these products, H. pylori VacA is a secreted toxin inducing vacuolation and apoptosis of epithelial cells. METHODS AND RESULTS: We found that non-opsonic human PMNL were sensitive to the vacuolating effect of VacA+ broth culture filtrate (BCF) and of purified VacA toxin. PMNL incubated with VacA+ BCF showed Rab7-positive large intracytoplasmic vacuoles. PMNL preincubation with H. pylori BCF of different phenotypes dramatically potentialized the oxidative burst induced by zymosan, increased phagocytosis of opsonized fluorescent beads, and up-regulated CD11b cell surface expression, but independently of the BCF VacA phenotype. Moreover, by using purified VacA toxin we showed that vacuolation induced in PMNL did not modify the rate of spontaneous PMNL apoptosis measured by caspase 3 activity. CONCLUSIONS: Taken together, these data showed that human PMNL is a sensitive cell population to H. pylori VacA toxin. However, activation of PMNL (i.e., oxidative burst, phagocytosis, CD11b up-regulation) and PMNL apoptosis are not affected by VacA, raising question about the role of VacA toxin on PMNL in vivo.     

Visible-Light-Induced Bactericidal Activity of a Nitrogen-Doped Titanium Photocatalyst against Human Pathogens

The antibacterial activity of photocatalytic titanium dioxide (TiO₂) substrates is induced primarily by UV light irradiation. Recently, nitrogen- and carbon-doped TiO₂ substrates were shown to exhibit photocatalytic activities under visible-light illumination. Their antibacterial activity, however, remains to be quantified. In this study, we demonstrated that nitrogen-doped TiO₂ substrates have superior visible-light-induced bactericidal activity against Escherichia coli compared to pure TiO₂ and carbon-doped TiO₂ substrates. We also found that protein- and light-absorbing contaminants partially reduce the bactericidal activity of nitrogen-doped TiO₂ substrates due to their light-shielding effects. In the pathogen-killing experiment, a significantly higher proportion of all tested pathogens, including Shigella flexneri, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Streptococcus pyogenes, and Acinetobacter baumannii, were killed by visible-light-illuminated nitrogen-doped TiO₂ substrates than by pure TiO₂ substrates. These findings suggest that nitrogen-doped TiO₂ has potential application in the development of alternative disinfectants for environmental and medical usages.