Metronidazole resistance of Helicobacter pylori is unstable

M2048

mutant strain. No motility on semisolid medium was also observed in mutant strain. Tire colonization density of H. priori in BALB/cmice by mutant strain on the 1~ week and by r-FlgK immunization on the 4'~ week were less than those of wild-type strain (P<0.05). Conclusions: FIgK plays an important role in the flageIla formation and H pylon motility. Deficient FlgK in mutant or by immunization with r-FlgK can decrease the colonization of H. pylori. FlgK can be a novel target promising for vaccination of H. pylori infection

Relationship between the Serological Response to H. pylori and the Morphological Variables of Antral Gastritis in Patients Infected by CagA Positive and CagA Negative Strains Michele 5ozzi Pao]o De Paoli, Rosamaria "l'edeschi, Renato "falamini, Natale Figura, Guillermo Perez-Perez, Amonino Carbone Background and aim Gastric inflammation, atrophy and intestinal metaplasia are motv severe

M2051

in H. pylo~i+ subjects, particularly in those cawing CagA + strains, than in controls. Since immunity can be involved in the development and persistence of chronic gastritis, our aim was to assess, in a group of H. pylor~+ patients, whether the antibody levels to H. pylori and CagA antigens are related with the histological scorns of antral gastritis. Material and Methods Serum samples and gastric antral biopsies were taken from 70 H. pylonS+ patients. H pyIo*J and CagA lgG antibodies were tested by standardized ELISAs. CagA status was also assessed by Westeru blot. Gastritis was classified according to the Sydney System. A score from 0 to 3 was assigtmd to each of the fullowing variables: atrophy, intestinal metaplasia, inflammation, and activity. The associations between IgG titres and histological katures were assessed by' means of chi-square test. Results FiftDone of the 70 patients were carrying CagA+ stratus. Associations between the levels of H D,lori antibodies and atrophy (p=0.03) and intestinal n~etaplasia scm~.'s(p=O.02) were found in the H. pylori+ and in the C~gA+ groups but not m the CagA- group. When the analysis was stratified according to age groups, these associations did not change. No associations were fuund between the leveg of H. pylori antibodies and the scores of inflammation and activity. In the H. pylon + and H. pylo~ +/CagA + groups, where associations between H. priori antibodies and atrophy and intestinal metaplasia were present, no asscx:tations were, however, found between these histological variables and the kvels of Cage4 antibodies. Conclusions. In subjects with H. pylori CagA + strains the semlugical response to the whole-cell antigens of the microorganism, but not to the CagA protein, seems to be related to the degree of gastric atrophy and intestinal metaplasia. We hy'pothesize di.at this phenomenon is related to antigenic mimicry between gastric epilhehat ceils and still unknown H.pylori antlgens co-expressed with the Cage4 protein

Metronidazole Resistance of Helicobacter pylm~ Is Unstable Qunaheng Song, Janet Pruckler, Patti Fields, Bala Swaminathan, Benjamin D. Gold The prevalence of H. pylori metronidazole (MTZ) resistance varies from 20% to 90% and is dependent on geographic region and population demographics. However, previous studies demonstrated MTZ resistance might not be stable, and may not correlate with therapeutic outcomes. The aim of this study ",','as to investigate whether MTZ resistance in H. pylori isolates was stable after > 1 year storage at -70 ~ Methods. H. pylori isolates were prospectively collected by the Centers for Disease Control and Prevention (CDC) from multiple sites across the USA in the Hdicobacter pylori Antimicrobial Resistance Project (HARP). H. pylori were isolated from gastric biopsies and identified using biochemical and PCR assays. Antimicmbial susceptibility was tested once the isolates amved at CDC using the NCCLSstandardized afar dilution method. After imtial testing, isolates were stored at -70 ~ After 1 + year of frozen storage, susceptibility testing was repeated (median storage time 2 years, range 14 years). ATCC type strain 43504 was used as the quality control strain in both initial and repeat susceptibility, testing. The breakpoints of the amimmmbial resistance used in this study are shown in Table 1 Results. Of 378 isolates, 53 isolates determined to be resistant to MTZ (MIC-->32) in the original tests were re-tested as described. Among tbese 53 isolates, 16 (30%) isolates reverted to MTZ sensitive (M1C --- 4.0). The MTZ MIC of the quality control strain remained stable over the study, period. Eleven isolates determined to be resistant to clarithmmycin in original tests were also re-tested. All of these strains (11/ 11) were still resistant, and 91% (10/11) were found to have a 165 rDNA mutation using a real-time PCR, indicative of clarithromycin resistance. Conclusion. Our data provide additional evidence for instability of MTZ resistance in H. pylori stratus. Conversely-,H. pylon clarithromycin resistance appears to be stable. Outcome studies comparing in vitro resistance to success/failure of eradication in MTZ resistant prevalent and susceptible geographic regions/populations are needed to determine the overall impact of MTZ resistance in determining effective treatment strategies.

M2049

Inhibition of iNOS by Polyamines Leads to H. pylori Survival Alain P. Gobert, Yulan Cheng, Darren R Bhimberg, Francoise i. Bussiere, Hangxiu Xu, Rupesh Chaturvedi, Keith T Wilson

Table I. breakpointe of H. py/od antimicrobial resistance (mglt)

BACKGROUND: We have sbown that when cultured with macrophages, H. pylori (Hp) 1) induces iNOS 2) is killed hy NO, and 3) induces arginase II and omithine decarboxylase (ODC) the enzymes responsible for conversion of arginine to omithine, and synthesis of polyamines, respectively. ODC converts ornithine to the polyamine putrescme, which is converted to spermidine and then spermine Spermidine and spennine, but not putmscine, are oxidized to torm aldehydes Spermine inhibits NO production in LPS-stinnalated macrophages, but the mechanism has not been determined. AIM: To determine if spermine and other polyamines inhibit iNOS in tip-stimulated macrophages, the mechanism underlying this effect, and the impact on Hp survival. METHODS: Hp SS1 was cultured with RAW 264.7 nlacmphages in the presence and absence of polyamines. Concentrations of the NO metabolite, NO2, were measured by Griess assay iNOS mRNA and protein levels were measured by RT-PCR and Western blotting, respectively. Hp grow,th was quantified by serial dilution and culture RESULTS:Hp bacteria or lysates stimulated a 7-fold increase in N Q production by macrophages at 24 h The increase m NO2 ,,`"asinhibited in a concentrationdependent manner by 50% (p < 0.05) to 100% (p < 0.001) with 1 - 5 ~M spermine. There was a parallel inhihitory eflect on iNOS protein levels. Similar inhibition was observed with sperrnidine, but not with lmtrescine. When assessed at 6 - 24 h after Hp stimulation, iNOS mRNA levels were markedly upregulated at each ume point, while iNOS protein was ~wt detectable until 18 h and peaked at 24 h. Spermine had m) eflect on iNOS mRNA levels, but prevented iNOS protein detection at 18 h and reduced the level by 85% at 24 h (p < 0.001). When macrophages were co-cuhured with Hp for 24 h to induce iNOS, washed, and then incubated with spemline, there was no loss of iNOS protein at 48 h, indicating that spermine does not decrease iNOS levels by causing protein degradation. Alter 24 h, Hp added alone to DMEM culture medmm increased r-ore 3.4 x 107 to 6.9 x 10e CFU/mL Co
Antiminrobial agent C,l a d t l i ~ n M~eonida~le

Sensitive 025 8,0

Intermediate 0,5 16

Resistant l.O 32

M2052 Presence and Characterization of Tetracycline Resistant Helicobacter pylori in USA Jeng Yih Wu, Jae J. Kim, Miae Lee, Mototsugu Kato, Rita Reddy, David Y. Graham, Dong H. Kwon Background: Successful treatment of H. priori infection heals or prevents the H. pflori related-diseases: gastric ulcer, duodenal ulcer, atrophic gastritis, gastric adenocarcinom& and MALToma. The presence of antibiotic resistant H. pylori has become the major impediment preventing successful therapy. Tetracycline is commonly used as a component of eradication therapies. Tetracycline resistant H. pylori have reported in Asia and Europe. Tetracydine resistant H. pylori have been proposed to be related to point mutations on tetracycline binding pocket (AGA~65.967)of the gene encoding 165 rRNA Aim: To identify, if tetracTcline resistant H. pylori are present m the US and, if so, the mechanism(s) of resistance. Methods: H. pylori collected between 1999 and 2000 from the US `'vere tested using agar dilution MIC measurements. Tetracycline resistant H. pylori are defined as MIC -> 4 m g / L of tetracycline. Results: 10 tetracycline resistant H. pylorr were tbund in 9 of 255 (3.5%) patients. 8 strains had low- or moderate-level resistance (MIC 4 to 16 mgfl_) and 2 strains had highlevel resistance (MIC >32 mg~) to tetracycline. Randomly amplified polymorphic DNA (RAPD)-PCR fingerprinting analysis revealed these 9 patients were infected with dilterent strains. One of the 9 patients was infected with two different tetracycline resistant strains. DNA sequence analysis of the tetracycline binding pocket of the 16S rRNA encoding gene showed that the 8 modemtedevel tetracycline resistant strains exhibited one of 4 different types (i.e., tGA, AGc, AGt, gGA) of point mutations on the AG&~,50~,r.The 2 high-level tetracycline resistant strains exhibited no changes in the tetracycline binding pocket. Conclusion: Tetracycline resistant H. pylori are present in the US Low- or moderatedevel tetracycline resistant H. pylori appear to be due m point mutations on AG&6~9~7of the 16S rRNA gene. Ho`'vever,no change in the tetracycline binding pocket among high-level tetracycline resistam H. pylor/ suggests presence of an additional rnechanism(s) tbr the high-level tetracycline resistant H. pylon.

M2050 Immunization with r-FlgK Achieves a Novel Role to Diminish the Colonization o[Helicobacter pylori in BALB/c Mice Bor-Shyang Shen, Jiunn0ong Wu, Ay-Huey Hnang, Hsiao-Bai Yang Backgruund/Aims: In H. pykm, fl~L4 and [lab are two major flagellin genes required for motility in stomach However, other flagellar structure proteins are not well understood. This study aimed to determine the hinctional actMty in vitro and in vivo offlgK, and to test whether flgK couId be sepeed as a target for vaccine immunization Methods: PCR ckming was u~ed to identity the flgk gene in H. pyIo*~isolates. Isogenoic flgK mutant was successfully constructed by gene replacement and confirmed by Southern blotting analysis. Flagellar filaments of H. pylor~ were demonstrated by electron microscopy (EM). Motility test was ob~rved m semisolid medimn The recumbent-FlgK protein was purified for immunization. H. pylor~ colonization density in the specific pathogen free BALB/cmice after 3-day 108 CFU inoculations wen: compared among wild, flgK mutant, and immunization with r-FlaK Resuhs: jlgK gene is conhrmed to contained 1,821 bp, predicted 606 amino acids, and has 957% identity widi published ~quences. tinder EM, no flagella filamem was observed in

M2053 High Frequency of Clarithromycin and Metronidazole Resistance in Helicobacter pylori isolates from Alaska Natives Michael Bruce, Brian McMahon, Julie Morris, Alisa Reasonover. Tom Hennessy, Debra Parks, Alan Parkinson Background: H. pylori infection is common (75% seroprevalence of IgG antibodies) among Alaska Natives (AN). In this population, it is a major cause of gastritis and peptic ulcer disease, and may be associated with gastric cancer rates that are over three times the national as,erage. Elsewhere in the U.S., H. pylori cure rates are high (85-95%), reinfection rates are low (< 5% over a 2-year period) and overan resistance to metronidazole (MtzR) and clarithromycin (ClaP.) varies from 35.1-38.7% and 9.1-11.1%, respectively. Both MtzR and

A-405

AGA Abstracts