Yukie Okimura

In Vitro Activities of Rabeprazole, a Novel Proton Pump Inhibitor, and Its Thioether Derivative Alone and in Combination with Other Antimicrobials against Recent Clinical Isolates of Helicobacter pylori

ABSTRACT The MICs of rabeprazole sodium (RPZ), a newly developed benzimidazole proton pump inhibitor (PPI), against 133 clinicalHelicobacter pylori strains revealed a higher degree of activity than the another two PPIs, lansoprazole and omeprazole. Time-kill curve assays of RPZ, when combined with amoxicillin, clarithromycin, or metronidazole, disclosed that synergistic effects were demonstrated in combination with each antibiotic examined. Moreover, no apparent antagonistic effect appeared among all of the strains tested.

Serum immunoglobulin G immune response to Helicobacter pylori antigens in Mongolian gerbils.

ABSTRACT The Mongolian gerbil model for Helicobacter pyloriinfection is an animal model that mimics human disease. We examined the serum immune response to H. pylori infection in gerbils by enzyme-linked immunosorbent assay (ELISA) and Western blotting, both with whole-cell (H. pylori) extracts. A total of 66 7-week-old specific-pathogen-free male gerbils were inoculated orogastrically with H. pylori strain ATCC 43504. Sera were collected 1, 2, 4, 8, 12, 26, 38, and 52 weeks after H. pylori inoculation. Sixty-nine noninfected gerbils and their sera were used as controls. The specificity of the ELISA was 95.7%. The frequency of seropositivity increased over time: 2 of 10 (20%), 7 of 10 (70%), and 7 of 7 (100%) samples of sera from inoculated gerbils were positive for H. pylori at 2, 4, and 8 weeks postinoculation, respectively. Western blot assays showed that the primary immunoglobulin G (IgG) response against low-molecular-mass (25-, 30-, and 20-kDa) proteins appeared after a lag period of 2 to 8 weeks after inoculation. Antibodies against 160-, 150-, 110-, 120-, 80-, 66-, and 63-kDa proteins were observed 12 weeks after inoculation. The early reactive 30-kDa protein was identified as a urease α subunit by N-terminal amino acid sequencing. After 26 weeks, two groups of animals could be distinguished: one group developed ulcers (n = 5), and the other developed hyperplastic polyps without ulcers (n = 19). Gerbils in the gastric ulcer group showed significantly higher serum anti-H. pylori IgG levels than did gerbils in the hyperplastic group (P = 0.001) as measured by ELISA. Furthermore, a higher proportion of animals developed antibodies to H. pylori proteins of 26, 25, and 20 kDa in the ulcer group than those animals with hyperplastic polyps (75 to 100% versus 17 to 50%) in Western blot assays. These results highlight the importance of the immune response of the host in the development of H. pylori-related gastric lesions.

Suppressive effect of rice extract on Helicobacter pylori infection in a Mongolian gerbil model

BackgroundRice extract has been shown to protect gastric mucosa from stress-induced damage. In this study, the antibiotic effect and the anti-inflammatory effect of orally administered aqueous rice extract on Helicobacter pylori infection and H. pylori-induced gastritis, respectively, in Mongolian gerbils were investigated.MethodsFifty specific-pathogen-free male Mongolian gerbils, seven weeks old, were divided into four groups: uninfected, untreated animals (group A); uninfected, rice extract-treated animals (group B); H. pylori-infected, untreated animals (group C); and H. pylori-infected, rice extract-treated animals (group D). Group C and D animals were killed 12 weeks after H. pylori infection (i.e., at 19 weeks of age) and group A and B animals were also killed at age 19 weeks. The stomachs were removed for histopathological examination with hematoxylin-and-eosin staining and anti-5′-bromo-2′-deoxyuridine (BrdU) immunostaining, and to determine the bacterial burden. Serum anti-H. pylori antibody titers were also tested.ResultsIn groups A and B, the gastric mucosa showed no inflammatory cell infiltration and a few BrdU-reactive cells. Group C animals developed marked chronic active gastritis in the gastric mucosa, and BrdU-labeled cells in the gastric mucosa markedly increased in number. In group D animals, a significant reduction occurred in the degree of neutrophilic polymorphonuclear cell infiltration into the gastric mucosa, in the BrdU-labeling indices of gastric epithelial cells, and in anti-H. pylori antibody titers in the serum (P < 0.01), compared with although H. pylori was not completely eradicated.ConclusionsThe rice extract was effective in suppressing inflammation and epithelial cell proliferation in the gastric mucosa in H. pylori-infected Mongolian gerbils. The rice extract has potential to exhibit a protective effect on H. pylori-related gastric mucosal diseases.

Successful Development of Air‐Dried Microplates (HP‐Plates) for Susceptibility Testing against Helicobacter pylori Isolates

We have successfully developed and evaluated a new susceptibility testing procedure against Helicobacter pylori strains using air‐dried microplates “HP‐Plates” containing eight serially‐diluted anti‐H. pylori agents. HP‐Plate wells were reconstituted by the inoculation of 100 μl of H. pylori cell suspensions. After incubation at 37 C for 48 hr under humidified microaerophilic conditions, HP‐Plates were read visually with a circular mirror. We investigated the within‐day reproducibility tests of HP‐Plates using the six quality control (QC) strains we proposed. Of the 20 testings, determining the minimum inhibitory concentrations (MICs) of all the QC strains fell within ± 1 log2 dilution ranges. When 200 clinical isolates were tested with HP‐Plates and compared with the results obtained with the modified broth macrodilution method of NCCLS, more than 90% of the MICs also fell within ±1 log2 dilution ranges. We concluded that the HP‐Plate susceptibility test method is a practical and easily applicable alternative of susceptibility testing for clinical microbiology laboratories in determining the MICs of H. pylori isolates.

Duodenogastric reflux and Helicobacter pylori infection synergistically increase gastric mucosal cell proliferative activity in mongolian gerbils

Background: Helicobacter pylori and duodenogastric reflux (DGR) are both recognized as aetiological factors in chronic gastritis and gastric carcinogenesis. In this study, a Mongolian gerbil (MG) model was used to investigate the histopathological changes in the gastric mucosa resulting from DGR and/or H. pylori infection. Methods: One-hundred-and-eleven 7-week-old, specific-pathogen-free, male MGs were divided into four groups: normal controls, gerbils with surgically induced DGR, and H. pylori-infected gerbils with and without DGR. Gerbils were killed 4, 12 and 26 weeks after DGR surgery, their stomachs removed and sections prepared. Sections were fixed immediately in 20% phosphate-buffered formalin and subjected to haematoxylin and eosin staining, Alcian blue at pH 2.5/periodic acid-Schiff staining, and immunostaining for smooth muscle cells, H. pylori and 5′-bromo-2′-deoxyuridine (BrdU). Results: The gastric mucosa of H. pylori-infected gerbils showed chronic active gastritis irrespective of DGR throughout the experimental period. The gastric mucosa of H. pylori-infected gerbils with DGR demonstrated higher BrdU labelling than in the other groups. Conclusions: In MGs, DGR and H. pylori infection synergistically increased gastric mucosal cell proliferative activity. DGR and H. pylori infection may be involved synergistically in gastric carcinogenesis by increasing cell proliferative activity.

In Vitro Activity of Cefotaxime,Ceftizoxime,Cefmenoxime,and Latamoxef in Comparison with Other β-Lactam Antibiotics against Recent Clinical Isolates of Haemophilus influenzae and Haemophilus parainfluenzae

The importance of Haemophilus injluenzae as an etiological agent of meningitis, septicemia and respiratory tract infection has been well documented for many years. Moreover, a number of infections due to H. parainjluenzae, such as septicemia, meningitis and endocarditis, have been reported (I, 2, 4, II, 12, 30). Ampicillin has long been considered the drug of choice for Haemophilus infections ( 18, 28). However, the emergence of strains resistant to ampicillin has recently been noted ( 5, 18, 23, 25, 26, 28), stimulating some investigators to study the in vitro efficacy of other antimicrobial agents against this species (3, 7, 9, 13-15, 21, 22, 29, 31). In the present study, we tested the in vitro activity of the newly developed semisynthetic third-generation cephalosporins, cefotaxime, ceftizoxime, cefmenoxime, and latamoxef, together with ampicillin, sulbenicillin, cefazolin, cefotiam, and cefoxitin, against recent clinical isolates of H. injluenzae and H. parainjluenzae. The 32 strains of H. injluenzae tested included 14 from pharyngeal swabs, 9 from sputa, 4 from ears, 2 from bronchial brushings, 2 from cerebrospinal fluid, and I from blood. All of the 68 H. parainjiuenzae strains examined were from pharyngeal swabs. The isolates were identified as H. injiuenzae or H. parainjiuenzae by testing for their requirement of X and V factors (16), for fermentation of glucose, sucrose, lactose, maltose, xylose, and mannitol (17), for indo! production (16), for urease activity ( 1 7), for ornithine decarboxylation ( 1 7), and for hemagglutination ( 16), and by the porphyrin test ( 19). All the strains were classifiable by biotype according to the proposal of Kilian (20). In addition, H. injiuenzae strains were serotyped with antisera types a to f (Difco) by using fresh agar cultures grown on chocolate agar plates containing 7.5% horse blood as the antigens. The presence of ,B-lactamase was assayed by the phenol red test as described by Escamilla (8). Minimal inhibitory concentrations (MICs) were determined by the agar plate dilution method. Chocolate agar plates containing the drug in twofold serial concentrations (from 100 to 0.00038 p,gfml) were prepared by adding 7.5% defib-

Prevalence and Biochemical Properties of Haemophilus Species in the Oral Cavity of Healthy Adults—Investigation of Three Japanese Individuals

Haemophilus species are now well known to inhabit the upper respiratory tract of healthy humans (9, 17). Until 1976 when Kilian (8) proposed the notion of biotypes in Haemophilus species, however, there was no real way of dividing the species into smaller groups. Moreover, recent studies have shown that Haemophilus species can be found at a high frequency in the oral cavity of humans (10, 11, 15). In Japan, however, little has been found out with regard to the occurrence and the biochemical proper tiesof oral Haemophilus species. For a correct understanding of infections due to these microorganisms in oral cavity, it seems significant to know their prevalence in the normal oral flora, and the present study was carried out to provide such information. The saliva materials examined were gathered in sterile dishes, and the speci mensof dental plaque were collected by scratching the teeth with sterile bamboo needles. Materials investigated were obtained from one male and two female healthy adults. Each specimen was immediately diluted with sterile physiological saline solution in a serial ten fold fashion, and spread on chocolate agar plates containing 7.5% defibrinated horse blood. The inoculated plates were incubated in candle jars at 35C for 48hr. The plates containing the dilution yielding the largest number of colonies that were sufficiently distinct to allow accurate counting were selected, and the total number of the Haemophilus species was calculated from the number of colonies grown on the respective plates. Each colony on the chosen chocolate agar plates from each of three saliva and three dental plaque samples randomly selected in advance was subcultured for fur therexaminations, and compared with the isolates from normal pharyngeal flora and those of clinical origin previously described (4, 5). Their morphological characteristics were examined with Grams stain. Requirement of Xand/or Vfactor was tested using X-, V-multi-disks (Eiken Chemical Co., Ltd., Tokyo). Dependence of the X-factor was ensured by the porphyrin test of Kilian (7). He magglutination,the hemolytic activity, and the production of catalase and H2S were examined as described previously (4). In addition, acid production from