Lower concentrations of clarithromycin suppress urease activity, motility, and binding to gastric epithelial cells in Helicobacter pylori isolates

ALIMENTARYTRACT

DICESTLIYEA OIS 2002;34:489-97

Lower concentrations of clarithromycin suppress urease activity, motility, and binding to gastric epithelial cells in tielicobacter pylori isolates K. K. M. K. T. T. K.

Nobata Ina Ohtal Kawamura-Satol Tsuzuki Ando Kusugami

Background.

Our previous

study showed

that histological

scores

of gas-

tric mucosal inflammation and Helicobacter pylori density decreased even in patients who failed to eradicate Helicobacter pylori after antimicrobial therapy including clarithromycin. This may reflect indirect suppressive effects of lower concentrations of clarithromycin on Heli-

cobacter pylori, as suggested in other Gram-negative rod infections. Aims. To investigate whether clarithromycin suppresses virulence factors of Helicobacter pylori at sub-minimal inhibitory concentration. Methods. Six clarithromycin-susceptible Helicobacter pylori isolates and 7 clarithromycin-resistant isolates were obtained from patients with peptic ulcer disease. These isolates were analysed for urease activiv, motility, and ability to bind to gastric epithelial cells after they were incubated with or without clarithromycin at sub-minimal inhibito-

ry concentrations. Results. Incubation of Helicobacter at sub-minimal inhibitory concentrations iv, and binding These findings

to gastric epithelial were observed both

pylori isolates

with clarithromycin

reduced urease activity, motilcells in a dose-dependent manner. in clarithromycin-susceptible and -

resistant strains. Conclusions. Suppressive effects of clerithromycin on virulence factors of Helicobacter pylori at sub-minimal inhibitory concentrations may be associated with observed attenuation of gastric inflammation and Helicobacter pylori density in patients who failed in bacterial eradication after triple therapy including clarithromycin. Fmw First Department uf Internal Medicine; 2 Department of Ba~l&7logy,Nagoya University School of Medicine, Nagoya, Japan.

Digest

Liver

Key words:

Ois 2002;34:489-97 clarithromycin;

Helicobacter py/ori; virulence

factor

Introduction Dr. K. Kusugami, First Department of Internal Medicine, Negoya University School of Medicine, 65 Tsurum&chv, Show&u, Nagaye 466-8550, Japan. Fax: &I-52-742157. E-mail: [email protected]. Authors tink Prof: H. Saito (First Depkof Internal Medicine, Nagoya UniversiLy School of Medicine, Nagoya, Jepan? far suggpstions end encouragement throughout study. Submitted Jury 4, 2001. Accepted after i&ion March 20, 2002.

It has become clear that Helicobacterpylori (H. pylori) is the major pathogenic factor in peptic ulcer disease (PUD) and eradication of this organism results in a dramatic change in the disease history lm4.The peptic ulcer (PU) recurrence rate decreases to O-10% in patients in whom H. pylori infection is cured, compared with an ulcer relapse rate of 33-95% in patients with ongoing infection 5. Such benefits derived from H. pylori eradication prompted the clinical investigators to accomplish better treatment regimens. Triple therapy, consisting of a proton pump inhibitor and two selections of antimicrobial drugs in various combinations, is currently recommended for H. pylori-positive patients with PUD from the standpoint of higher efficacy, fewer side effects and better patient compliance 6 ‘. Among antimicrobial drugs, clarithromycin (CAM), an acid stable macrolide antibiotic, has been widely used to cure H. pylori infection because it shows a low minimal inhibitory concentration (ME) for H. py489

Clarithromycin

and H. pyhri

lori with an ability to induce a good diffusion in the gastric mucosa 8. The resistance of H. pylori to CAM is, therefore, an important factor influencing the treatment outcome when we treat H. pylori-positive patients with PU, as indicated by the studies showing that the eradication rate in patients with CAM-susceptible (CAM-S) H. pylori is higher than that in patients with a CAM-resistant (CAM-R) organism 8m’0.On the contrary, further evidence suggests that primary resistance to CAM could be overcome by a triple therapy including CAM ‘I, suggesting that CAM might have certain suppressive effects on H. pylori other than the inhibitory activity for bacterial growth. Macrolide antibiotics have been shown to inhibit bacterial virulence factors at sub-MICs in various Gramnegative microorganisms ‘Z-14. For example, lower concentrations of macrolide antibiotics, including CAM, could inhibit motility and protein synthesis in Pseudomonas aeruginosa (19 aeruginosa) without affecting bacterial growth, and this provides a basis for explanation why long-term administration of lowdoses of macrolides could improve the clinical symptoms and prognosis of patients with chronic respiratory infections with P. aeruginosu 14-“. Our previous study showed that histological scores of gastric mucosal inflammation and H. pylori density decreased even in patients who failed to eradicate H. pylori after antimicrobial therapy including CAM although these phenomena were less prominent than in those who succeeded in eradication I8 19. In consideration of the observation that MIC of CAM, for most resistant strains, is in the range that is held to be substantially higher than the concentration achieved in the gastric mucus after oral administration 8 *‘, it is possible that CAM is able to inhibit virulence factors expressed by H. pylori even at lower concentrations without exerting bacterial growth inhibition. To address this hypothesis, we investigated whether lower concentrations of CAM could affect the prime virulence factors of H. pylori, such as urease activity, motility and binding to gastric epithelial cells, using CAM-S and CAM-R strains.

Materials and methods Patients and H. pylori isolates A total of 150 H. pylori-positive patients with PUD (gastric ulcer, n=7 1; duodenal ulcer, n=79) were treated for one week with a combination of omeprazole, 20 mg twice daily, CAM, 400 mg twice daily, and amoxycillin (AMPC), 750 mg twice daily or a combination of omeprazole, 20 mg twice daily, CAM, 400 mg twice daily, and tinidazole, 500 mg twice daily. Eradication was diagnosed in 135 patients (90%) according to the ab490

sence of H. pylori, at 6 months after therapy, in all of four assays: bacterial culture, CL0 test (Delta West, Bentley, Australia), identification of the organism in tissue sections stained with Giemsa, and ‘“C-urea breath test 18. Prior to treatment, H. pylori strains were isolated from endoscopic antral biopsies, as previously described 2’. In total, bacterial isolates were obtained from 122 patients in whom H. pylori was successfully eradicated (success group) and 13 in whom H. pyZori eradication failed (failure group). Antimicrobial susceptibility tests for CAM were conducted in 40 H. pylori isolates from the success group and all 13 from the failure group using an agar dilution method *“. In the former group, all H. pylori isolates were found to be CAM-S strains (MIC,
K. Nobata et al.

broth were transferred in an aliquot of 1 ml to 4 ml of fresh broth with or without various concentrations of CAM or AMPC and cultured at 37°C under microaerobic conditions for 12 hours. At the end of the culture period, the number of bacteria was counted by optical density (OD) measurements (A560). A constant number of bacteria (1~10~ cells) were suspended in distilled water and disrupted by sonication. The supernatant obtained after centrifugation (10,000 g, 20 minutes, 4°C) was used for the urease assay. The supernatant of bacterial lysate (100 ul) was added to 100 mM sodium phosphate buffer (pH 7.0) containing 100 mM urea (300 ul), and the reaction mixture was incubated at 37°C for 30 minutes, after which the reaction was terminated by addition of 100 ul of 1N H2S04. Then, 500 ul of solution containing 1% phenol-0.005% sodium and alkali reagents (5.5% Na2HP04, 0.5% NaOH, and 0.1% NaOCl) were added, and the mixture was incubated at 37°C for 40 minutes. The colour development was monitored at 540 nm. Urease activity in the supernatant of bacterial lysate was calculated by referring to a standard curve made by known concentrations of jack bean urease (Sigma, St. Louis, MO, USA). In this assay, one unit of urease released 1 uM of ammonia in the bacterial lysate per minute. Motility test Motility of H. pylori isolates was assessed by a single-colony motility assay, as previously described 25. In brief, bacteria (1~10~ cells) grown in brucellaserum were harvested in 1 ml of 0.9% NaCl and diluted 1: 103. The motility agar consisting of brucella broth and 0.4% Bacto agar was cooled to a temperature of about 40°C after which various concentrations of CAM and 10% FCS were added. Bacteria were diluted to a final dilution of 1: 1O6 in the motility agar and poured into the plates (8.0 ml per plate). As controls for each sample, CAM was omitted in the motility agar. The plates were then incubated at 37°C under microaerobic conditions for 7 days. Singlecolony morphology of H. pylori isolates was examined with a phase-contrast microscope at magnifications of x10-40. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) The flagella proteins of H. pylori isolates were prepared by the method of Montie et al 26. with a minor modification. Briefly, bacteria grown in brucellaserum broth (6 ml) were cultured at 37°C under microaerobic conditions for 12 hours after the addition of fresh broth (24 ml) with and without various concentrations of CAM. On completion of the culture, bacteria (3~10~ cells) were suspended in phosphate buffer saline (PBS) and then blended in a commercial

blender for 3 minutes to shear off the flagella. The suspension was centrifuged at 5,000 g and 4°C for 10 minutes and the resulting supernatant was centrifuged again at 40,000 g and 4°C for 3 hours. The supernatant was carefully removed and the pkllet was suspended in 10 yl of PBS. After the total protein concentrations in these samples were determined using a Micro BCA Protein Assay Reagent Kit (PIERCE, Rockford, IL, USA), a portion of the flagellin preparation corresponding to 100 ug of protein was boiled for 30 minutes in Laemmli buffer containing 0.1% pmercaptoethanol and separated by 10% SDS-PAGE. The separated proteins were visualized with Coomassie blue. Electron microscopy Bacteria grown in brucella-serum broth were transferred in an aliquot of 1 ml to 4 ml of fresh broth with or without various concentrations of CAM and cultured at 37°C under microaerobic conditions for 12 hours. At the end of the culture period, bacteria were suspended in 0.9% NaCl, applied to carbon-film on a copper grid, fixed in 1% glutaraldehyde (pH 7.0) for 1 minute, and negatively stained with 1% phosphotungstate (pH 7.0) for 5-10 seconds. The grids were then examined with a JEM-2000 EX electron microscope (JEOL, Tokyo, Japan) 2s27. Binding assay Bacteria grown in brucella-serum broth (1 ml) were cultured at 37°C under microaerobic conditions for 12 hours after the addition of fresh broth (4 ml) with and without various concentrations of CAM. On completion of the culture, bacteria were suspended in PBS. A human gastric cancer cell line, MKN45 (obtained from the Japanese Cancer Research Resources Bank, Tsukuba, Japan) was used for the assay determining the ability of H. pylori isolates to bind to gastric epithelial cells 28. Briefly, MKN45 cells were seeded on g-well chamber slide (Lab-Tek Munc, Naperville, IL, USA) and cultured overnight in a 5% CO2 incubator. Then, the chamber was washed with PBS and incubated with the bacterial suspension (H. pylori: MKN45 cells, 20: 1) at 37°C for 30 minutes. After removing free bacteria by washing with PBS, MKN45 cells were fixed with 2% paraformaldehyde for 10 minutes. The chamber was incubated with anti-H. pylori polyclonal antibody (Dako, Glostrup, Denmark; I:100 dilution) for 30 minutes, followed by incubation with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibody (Cappel Laboratories, West Chester, PA, USA; 1: 100 dilution) for 30 minutes. The number of adherent bacteria was counted for 20 cells in 10 random fields under a BH-2 fluorescence microscope (Olympus, Tokyo, Japan). 491

Elarithromycin

and H. py/wi

Statistical analysis Statistical analyses were performed by Mann-Whitney U or paired t tests. Data were shown as the mean + SEM. A value of ~~0.05 was considered to be statistically significant.

growth in CAM-R isolates (Fig. la). The lysate of both CAM-S and -R isolates had substantial amounts of urease activity, however, no significant difference was observed between these two groups of bacteria obtained after incubation without CAM (CAM-S, 446k13/10s bacteria; CAM-R, 373+48/108 bacteria). Incubation of H. pylori isolates with CAM reduced

Results CAM resistance in H. pylori isolates Table I summarizes the clinical and bacterial profiles of 6 CAM-S and 7 CAM-R H. pylori isolates used in the present study. All 7 CAM-R isolates (MIC, >l ug/ml) possessed A to G point mutations at either position 2143 (n=l) or 2144 (n=6) in domain V of the 23s rRNA gene, while none of the 6 CAM-S isolates had these mutations as determined by PCR and direct sequencing, confirming earlier reports showing that point mutations at these portions are associated with resistance to CAM 22. By contrast, all these 13 H. pylori isolates were found to be susceptible to AMPC (MIC, co.05 ug/ml) in the antimicrobial susceptibility test using an agar dilution method. Effects of CAM on growth and urease activity in H. pylori isolates As could be expected from the results on MIC, a significant concentration-dependent suppression was observed for bacterial growth in CAM-S H. pylori isolates when they were incubated with CAM at concentrations of more than 0.03 ug/ml, where incubation with CAM yielded less significant inhibitions of Table I. Clinical and bacterial profiles of Helicobacter used in study.

Isolate NlJHP582 T56 T71 T72 T59 T257 NUHP629 TBIO NUHP453 T876 ND112 NUHP535 T14 Abbreviations:

492

py/ori

CMhwmycin WC &3/rnll

233 rRNA domain U

Ermlication -WY

DU GU GU GU DU DU DU DU GU DU DU DU GU

0.06 61 0.03 61 0.025 El <0.015 61 <0.015 6) <0.015 [Sl 32 [RI 16 [RI 8 (RI 8 m 8 (RI 4 [RI 4 (RI

Wild type Wild type Wild type Wild type Wild type Wild type 2143, A to 2144, A to 2144, A to 2144, A to 2144, A to 2144, A to 2144, A to

Success Success Success Success Failure Failure Failure Failure Failure Failure Failure Failure Failure

see list.

,

0

I

0.0006

I

a002

I

0.0075

I

I

am

~12

I

I

0.5

2

CAM ConGentratlon(p#mJ)

A

isolates

Dismme

G G G G G G G

0’

0’

(

0 B

I

0.0063

I

wb3iuo75

I

I

I

0.08

a12

I

I

as

2

~~-~bJimJ)

Fii. 1. Effects of CAM on growth [Al and urease activity fB1 in CAMS k-r=61 and CAM-R H. pylori isolates fn=7). 00 of 0.01 corresponds to bacterial concentration of 1x107/ml. * p
K. Nobata et al.

urease activity in the lysate in a concentration-dependent manner in both CAM-S and CAM-R strains, and statistically significant differences were detected at concentrations more than 0.0075 ug/ml (Fig. lb). Notably, a decrease in urease activity caused by CAM was already evident in the concentration range (0.0075 ug/ml) where bacterial growth was not inhibited. In these experimental conditions, the recovery of viable CAM-S and CAM-R H. pylori isolates, which was determined as colony forming units by an agar dilution method, was not affected by sub-MICs (0.002 and 0.0075 ug/ml for CAM-S isolates and 0.002, 0.0075 and 0.03 ug/ml for CAM-R isolates) of CAM (data not shown). By contrast, incubation of CAM-R H. pylori isolates with AMPC failed to decrease urease activity in the bacterial lysate, despite the fact that this antimicrobial drug showed a dose-dependent suppressive effect on bacterial growth (data not shown). Effects of CAM on motility in H. pylori isolates Motility of H. pylori isolates was assessed with a phase-contrast microscope by comparing the size of the swarming halos around a single colony in the motility agar. In the absence of CAM, H. pylori isolates seemed to be actively motile because they showed a granular colony with a surrounding swarming zone (Fig. 2a), which contrasted with a colony morphology with sharp dense outlines but without a significant swarming zone in isolates incubated with CAM at concentrations of more than 0.0075 ug/ml (Fig. 2b). This inhibition of motility caused by CAM was not influenced by whether the isolates were CAM-S or CAM-R. To check the flagelin expression, we performed SDSPAGE analysis of the flagella preparations and electron microscopic examination using CAM-R H. pylori iso-

Fig. 2. Effects of CAM on motility in CAM-R la&s. Abbreviations: see list.

pyhri isolates. (Al CAIN

Fig. 3. Effects of CAM on ultrastructure of CAM-R H. py/ori isolates as defined by transmission electron microscopy. [A) CAM, II @/ml; [El) CAM, 0.03 &ml. Bar, 500 nm. Similar results were obtained in CAM-S isolates. Abbreviations: see list.

lates. The intensity of the band for the flagellin (53-54 kDa) in H. pylori isolates obtained after incubation with CAM (0.002-o. 12 ug/ml) was similar to that detected in isolates incubated in the absence of CAM (data not shown). Similarly, the transmission microscopic examination showed that the morphology of the flagella in H. pylori isolates remained unchanged even after they were incubated with CAM at the concentration of 0.03 ug/ml that yielded a significant inhibition of bacterial motility in the single-colony assay (Fig. 3). On the contrary, H. pylori isolates incubated with the above concentrations of CAM seemed to be intertwisted with each other by coiled flagella in the scanning micro-

&ml;

[BI CAM, 0.03 &ml.

Similar results were obtained in CAM-S iso-

493

Clarithromycin

and H. pykwi

Fig. 4. Effects of CAM on ultrastructure of CAM-R H. py/afiisolates as defined by scanning electron microscopy. (Al CAM, 0 &ml; @/ml. Bar, 5 pm. Similar results were obtained in CAM-S isolates. Abbreviations: see list.

scopic examination, contrasting with the observation obtained in untreated isolates (Fig. 4). Essentially results identical were obtained in SDS-PAGE analysis of the flagella preparations and electron microscopic examination when we used CAM-S isolates. Effects of CAM on binding to gastric epithelial cells by H. pylori isolates Fluorescence microscopy was used to investigate whether incubation with CAM could influence the

T

n CAM-S 0 CAM-RI

l-----l

I. 5. Effects of CAM on binding to gastric epithelial cell-derived MKN45 cells in CAM-S fn=61 and CAM-R In=71 H. py/wi isolates. * pcO.05 compared with value obtained in absence of CAM. Abbreviations: see list.

494

(91 CAM, 0.03 4

binding of H. pylori isolates to the gastric epithelial cell line. All H. pylori isolates were found to bind to MKN45 cells and there was no significant difference in the binding capacity as assessed by fluorescence microscopy between CAM-S (97-+17 bacteria/20 epithelial cells) and CAM-R isolates (92k9 bacteria/20 epithelial cells, Fig. 5). More important, incubation of H. pylori isolates with CAM reduced their binding to MKN 45 cells in a concentration-dependent manner, and statistically significant differences were found at concentrations of 0.0075 and 0.03 ug/ml. Again, this inhibition was similar in CAM-S and CAM-R strains.

Discussion The concept that H. pylori is an important pathogenic factor in PUD has led to the wide use of antibiotics to eradicate this organism. The high cure rates have been achieved with multiple antimicrobial treatment regimens that include CAM ’ il. Although CAM is one of the most effective and frequently used antibiotics for curing H. pylori infection, treatment failure sometimes occurs, and the resistance of H. pylori to CAM is considered to be its primary cause 9. The mechanism of CAM resistance in H. pylori infection is known to be based on point mutations in domain V of the 23s rRNA gene that prevent the binding of CAM to the 50s ribosomal subunits 222g30. In the present study, all seven CAM-R clinical isolates of Ii. pyEori obtained prior to treatment were found to have A to G mutations at positions 2143 or 2144 of the 23s rRNA gene, thus confirming earlier studies showing that mutations in the 23s rRNA are associated with CAM resistance in

K. Nobata et al.

vitro 29. Moreover, a high rate for the presence of CAM-R H. pylori among treatment failures indicates that the susceptibility or resistance of H. pylori to CAM is a major factor determining treatment outcome (eradication or failure) in PU patients 9. Besides the inhibitory activity for the growth of H. pyZori, CAM may have suppressive effects on bacterial virulence factors at sub-MICs, and these properties may make an impact on the host-parasite interaction even if H. pylori infection is not successfully eradicated, as suggested in other chronic Gram-negative infections 14-17.Therefore, we investigated whether lower concentrations of CAM could affect urease activity, motility, and adherence to gastric epithelial cells in H. pylori isolates since these factors allow prolonged colonisation and survival of the bacterium in the gastric mucosa”‘. The results in the present study clearly indicate that CAM has in vitro suppressive effects on both CAM-S and CAM-R H. pylori isolates in terms of the expression of bacterial virulence factors at clinically achievable concentrations of lower than 0.1 yglml. It has been shown that bacterial growth could be inhibited by higher concentrations of CAM even when H. pylori isolates are defined as CAM-R and this inhibition occurs with a difference between mutations at positions 2143 and 2144 in the 23s rRNA gene 3’. These findings suggest that the binding of CAM to the peptidyl-transferase loop of the 23s rRNA may not be an “all or none” phenomenon; CAM could bind to the ribosomal subunits in CAM-R H. pylori isolates, as speculated from the fact that no dominant mechanisms for CAM resistance other than the conformational changes in its binding site are currently known 2229j”. The major antimicrobial activity of CAM is attributable to inhibition of bacterial protein synthesis by binding to the ribosomal subunits. In this context, we found that sub-MICs of CAM also induce a decrease in the content of intracellular proteins in CAM-R H. pylori isolates (unpublished observation). The suppressive effects of sub-MICs of CAM observed on bacterial virulence factors may be related to inhibition of the synthesis of proteins that are involved in the expression of these factors. Incubation of H. pylori isolates with CAM, but not AMPC, caused a concentration-dependent decrease in urease activity, and this effect did not differ significantly between CAM-S and CAM-R bacterial strains. It was also noted that a decrease in urease activity caused by CAM was already evident in the concentration range where bacterial growth was not inhibited. Compared with other bacterial pathogens, H. pylori has larger amounts of urease on the surface of the organism as well as within the cytoplasm, and this enzyme plays a central role in the pathogenesis of H. pylot-i infection j’. Evidence from animal experiments

suggests the necessary role of urease in colonisation of in the gastric mucosal layer 32. In addition, urease may act as a factor inducing mucosal injury through the production of ammonia or inflammatory cytokines, such as interleukin-8 and tumour necrosis factor-a 3334. The finding that CAM could suppress in vitro urease activity exhibited by H. pylori isolates at sub-MICs, independently of the states of CAM resistance, may partly explain why histological scores of gastric mucosal inflammation and H. pylori density improve after antimicrobial therapy with CAM even in patients belonging to the eradication failure group I8 19. Similarly, treatment of both CAM-S and CMA-R H. pylori isolates with CAM at sub-MICs (0.002-0.03 ug/ml) resulted in inhibition of bacterial motility in the assay of the motility gel. This inhibition of bacterial motility was not due to suppression in the de novo synthesis of flagellin protein or alteration in the ultrastructure of flagella, as shown in the SDS-PAGE and transmission electron microscopy. On the contrary, H. pylori cells incubated with sub-MICs of CAM seemed to be intertwisted with each other by coiled flagella in scanning electron microscopy, thus suggesting that inhibition of bacterial motility induced by sub-MICs of CAM may be related to the effect on the physiological or functional activity of the flagella. Further detailed characterisation of the mechanisms involved in H. pylori motility, including clarification of the role of urease and regulatory molecules for the biosynthesis of other flagellar proteins, could lead to a better understanding of the mode of CAM action in this phenomenon 3s36. The binding capacity of H. pylori isolates to the gastric epithelial cell line was shown to decrease after incubation with CAM at sub-MICs with a concentration-dependent effect. Again, this effect was almost identical in CAM-S and CAM-R bacterial strains. Since H. pylori is viewed as a non-invasive bacterial pathogen 37, its binding capacity to epithelial cells could be regarded as an important factor causing host-parasite interaction and subsequent inflammatory responses in the gastric mucosa. The inhibitory effects of CAM on in vitro H. pylori binding to the epithelial cell line at clinically achievable concentrations suggest this antimicrobial agent may produce histological improvement in H. pylori-associated gastric mucosal inflammation, at least partly, by modulating its binding capacity to the epithelium in vivo I6 19. CAM and related macrolide antibiotics are known to share antimicrobial activity by binding to the ribosoma1 subunits. Azithromycin, erythromycin, and CAM at sub-MICs show similar suppressive effects on bacterial motility and flagellin expression in P. aeruginova and Proteus mirabilis 14. To the best of our knowledge, there are no data on the suppressive effects of macrolide antibiotics on the expression of virH. pylori

495

Clarithromycin

and H. pylori

ulence factors in H. pylori. Our preliminary experiments (unpublished observation) showed that azithromycin inhibits motility of H. pylori isolates at lower concentrations (co.02 ug/ml) than MICs (0.060.5 ug/ml). It is necessary to conduct further detailed studies to clarify whether macrolide antibiotics exert common suppressive effects on the virulence factor expression in H. pylori. Recently, Matsuoka et al 23. showed the co-colonisation of CAM-S and CAM-R H. pylori strains in the stomach, and they attributed the appearance of CAM-R strains to spontaneous point mutations in the region of the 23s rRNA gene. This implies that the standard antimicrobial susceptibility test may underestimate the frequency of CAM-R H. pylori strains since this method covers only a limited population of the bacteria colonised in the gastric mucosa. Nevertheless, multiple antimicrobial treatment regimens that include CAM have achieved high rates of H. pylori eradication, which could be partly explained by the suppressive effects of CAM on virulence factors expressed by both CAM-S and CAM-R bacterial strains, in addition to its antimicrobial activity against susceptible strains ’ ‘. Our data validate the use of CAM as a primary antimicrobial agent for H. pylori eradication when we consider the treatment for patients with PUD.

List of abbreviations AMPC: amoxycillin; CAM: clarithromycin; CAM-R: clarithromycin resistant; CAM-S: clarithromycin susceptible: FCS: foetal calf serum; H. py/ori: Hekobacter py/ori; MIC: minimal inhibitory concentration; GU: gastric ulcer: OD: optical density; P. aeruginosa: Pseudomonas aeruginosa; PBS: phosphate-buffered saline; PCR: polymerese chain reaction; PU: peptic ulcer; PUD: peptic ulcer disease; R: resistant; S: susceptible; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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3m WORLD CHINESE CONGRESS OF DIGESTOLOGY September 23-25,2002 The Third World Chinese Congress of Digestology (WCDD) will be co-sponsored by the World Journal of Gastroenterology (English), World Chinese Journal of Digestology, and Diagnosis and Treatment of Digestive Diseases, September 23-25, 2002 in China. Contact: Lian-Sheng Ma President of WCCD PO. Box 2345, Beijing 100230, China Fax: +89-65891893 l

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