Is epsilometer test (E-t) to determine metronidazole susceptibility (MS) of H. pylori (Hp) really unreliable?

ClaR have beeu associated with higher treatment failures Methods: We analyzed H. pylori data from CDC's sentinel surveillance in Alaska to determine: 1) the proportion of culture positive biopsy specunens from AN adults undergoing routine upper endoscopy, and 2) the susceptibility of H. pylori isolates to metronidazole, clarithromycin, tetracycline and amoxicillin We cultured clinical specimens obtained from biopsies of AN patients undergoing upper endoscopy from 7/99 through 7/02 at four medical t?tcilities in urban and rural Alaska. Susceptibilines were determined by agar dilution. Results: Of the 906 biopsy specimens obtained from 616 AN patients 444 (49%) were culture positive, representing 318 (52%) patients. A similar proportion of males and females were cuhure-posRive (54% vs 50% OR 1.2, p = 35). Mean age of both culture-positive and culture-negative patients was 51 years, 177 (43%) of 411 isolates demonstrated MtzR with a minimum inhibitor}' concentration (MIC) of > 8 g,g metronidazoledml, 136 (33%) isolates demonstrated ClaR with an MIC of > 1 ~g clanthromycin/ml, 7 (2%) isolates were resistant to amoxlcfllin with an MIC of > 1 ~g amoxicilhn/nfl and one isolate was resistant to tetracychne at 2/a,g tetracycline/ mL MtzR and ClaR varied by region (ranges, 39-63% and 31-44%, respectively), but not by urban vs, rural residence. Females were more likely than males to show MtzR (52% vs. 32%, OR = 2.3, p < .013; however, the same was not true for OaR (35% vs. 30%, OR = 1.3, p = .303. No difference in mean age was observed for MtzR (mean age resistant 48.6 yrs vs, mean age sensitive 5L8 yes, p = .083 or OaR (mean age resistant 50.7 yrs vs. mean age sensitive 50,2 yrs, p = .81). Conclusions: Rates of resistance to metronidazole and clarid:lromycm are higher among AN when compared with the rest of the Us A high prevalence of mt~:ctionwith H. pylori, combined vr a high Mquency of antibiotic resistance in H. pylon isdates could resuh in high rates of treatment tailure.

to transform (frequency of 1.5x10-5) susceptible H, priori 26695 to ~-lactam antibiotic resistant H. pylori (MIC 8 to 16 mg/L). Conclusion: Significant alterations in the mosaic block of pbp-IA may, produce high-level resistance to p-lactam antibiotics, The mosaic block can spread among clinical H. pylori isolates with acquisition of high-level !3-lactam antibiotic resistance. MIC values of the transformed ]3-1actamantibiotic resistant H. pylori were lower than those of the clinical resistant H, pylori, suggesting presence of an additional ]Mactam resistance mechanism(s) in the clinical resistant H. priori.

M2056 THE EFFECTIVENESS OF ANTIBIOTIC SUSCEPTIBILITYTEST BY DILUTION AGAR METHODS AND DETECTION OF POINTMUTATIONS IN THE 23S RIBOSOMAL RNA BEFORE SECOND-LINE TREATEMENT OF HELICOBACTER PYLORI INFECTION Tatauhiro Masaoka, Hidekazu Suzuki, Kumiko Kurabayashi, Akitoshi Kamiya, Hiromasa Ishii Back ground & Aim. Although, 7-day triple therapy with Iansoprazole (LPZ) or omeprazole (OMZ), amoxicillin (AMOX) and clarithromycin (CAM) is approved only for treatment of Helicobacter pylori(H.pylo~) infection m Japan, strains which cannnot be eradicated by this regimen are increasing. To select the most effectwe second-line treatment for H. pylori intection, we examined the antibiotic susceptibility by dilution agar methods and detection of point mutation in the 23S ribosomal RNA before second-line treatment for H. pytori. Methods. 43 patients with H. D'lori positive peptic ulcer or chronic gastritis after failed 7day"triple therapy' with LPZ, AMOX and CAM (LAC therapy) were allocated and underwent upper G1 endoscopy. Endoscopic biopsied specimens were used tbr examining antibiotic susceptibility to CAM, metronidazole (MET) and AMOX. We measured Minimum Inhibitor}' Concentration(MIC) of each antibiotics were defined by agar dilution methods. Since H pylori resistance to CAM is connected with a point mutation of the adenine at position 2143 or 2144 of 23S ribosomal RNA(Antimicrob. Agents and Chemotber. 40:477, 1996), we also studied strains whether they' had a point mutation or not by the Polymerase Chain Reactionrestriction fragment length polymorphism (PCR-RFLP) methods with Bsa[ and MboII restrictton enzymes. Resistance breakpoints were : (;AM I ~g/ml, MET 8 b~g/ml,AMOX > 0.03 t*g/mL Strains which acquired no drag I~sistance were eradicated by LAC therapy, again. Strains whmh were resistant to CAM and susceptible to MET were eradicated by 7-day triple therapy with LPZ, AMOX and MET (I_AMtherapy). Eradication was confirmed by a negative urea breath test 12 weeks after the completion of treatment. Results. The frequencies of resistance alter unsuccessful LAC therapy were: CAM, 72.0 % ; MET, 9.3 % ; AMOX, 16.3 % By diluuon agar methods, 6.5% of resistant strains which were digested by Bsal o r / ~ I I could not be detected. The rate of secondary eradication after the antibiotic susceptibility' test was 91.3 %. Conclusion. Dilution agar methods confirmed by PCR-RFLP methods can improve the diagnosis rate of resistance to CAM. Both methods should be performed to select the most effective tseamtent tbr H. pylod refection.

M2054 Are There Regional Differences in Clarithromycin Resistance in the United States? Results from an H pylori Eradication Trial with Rabeprazole Triple Therapy Nimish Vakil, ~rlos Perdomo, Jay Barth Purpose: Previous studies have suggested that clarithromycin resistance in the United States may be more common in the Northeast and less common in the Midwest, although data are itK-onsistent Clarithromycin sensitivity across US geographic regions was analyzed in a large efficacy trial of rabeprazole-based triple therapy fbr H pylori eradication Methods: This mukicenter, double-blind, controlled trial randomized 803 patients with confirmed H pylon infection Patmnts received 1 oI 4 eradication regimens: rabeprazole 20 mg bid, amoxiciUin I g bid, and danthromycin 500 mg bid for 3, 7, or 10 days (K4C-3, -7, and -103 or a control regimen of omeprazole 20 mg bid plus the same antibiotics for 10 days (OAC-IO), Before treatment, antintmrobial sensitivity testing was conducted on H pylor~ isolated from gastric biopsies Results of testing, available in 560 of 783 intent-to-treat (i273 patients, showed that 53 isolates were intermediate or resistant (2 intermediate; 51 resistant) to clarithmmycin. Results: Clarithromycin susceptibility" is showm in the table Logistic regression analysis tbund no a~ciation between regmn and sensitivity. Conclusions: Pretreatment clarithmmycin 1~sistance of H pylori was not asscx:iated with geographic region in this large US trial, Regional changes in eradication regimens are tbereIore not necessary in the Uuited States. ReseaTvhsupported by Eisai Inc. Teaneck, NJ and Janssen Pharmaceuttca inc., TitusviIle, NJ,

M2057 Is Epsilometer test (E-t) to determine metronidazole susceptibility (MS) of H, pylori (Hp) really"unreliable? Fedenco Perna, Luigi Gatta, Natale Figura, Chiara Ricci, Andrea Tampien, Mario Miglioli, Dmo Vaira

Pretreatment Clarlthromycin Suscept~lity by Geographic Region (Igl"Patients),n(%) Susce~bilRy

Northeast (n=68)

Sensitive (MIC<_~5 (M1r

61(90)

Midwest South (n=41) Atlantic (n=152)

Sooth Central (n=~5)

West (n=~4)

Total (N~)

39(95)

84(88)

186(91)

507(91)

1102)

18(9)

53(9}

137(90)

pg/mL) 7(10) 2(5) 15(10) (MIC..O ~mL) Logistic regression analysis: probaNily=0,8009. or R ~ n t

Background: the reliability of E-t in determining MS of Hp has recently been questioned with possible clinical implication in the management of patients who undergo Hp eradication. Aim: 13 to compare E-t and the disk diffusion (DD) to the agar dilution (AD), for determining the susceptibility of Hp to metronidazole (MZ); 23 to investigate whether potential discrepancies could be due to the simultaneous presence of MZS and MZR bacterial subpopulations. Methods: 109 Hp strains from 121 consecutive patients were examined. All tests were carried out starting fi'om primary plates. AD was per|brmed according to NCCLS, E-t according to manufacturers guidelines and DD using 5-mug MZ disks. Isolates were considered MZ-R if MIC was > 8 mug/ml for AD and E-t, or if the inhibition zone around the disk was <20 mm for DD. 43/109 isolates were also investigated to detect mixed infection: 100 ml of bacterial suspensions of each strain were seeded onto plain agar plates (PAP) and plates containing 8 mug/ml of MZ (MZP), Cultures were considered mixed if the number of colonies on PAP exceeded by at least 1-1oglO the one on MZP. Results: according to AD, 57 strains (52.3%; 95%CI:43 to 61 4) were MZ-R. E-t misdiagnosed 2 strains (considered MZ-R) whilst DD misdiagnosed 3 strains (2 MZ-R and 1 MZ4). Discordance was found 1,9% (95%CI:0,5 to 6.6), and in 2.8% (95%CI: 0.9 to 7.8) respectively when E-test and DD were compared to AD. Imer-test variability among AD and E-test showed that 39.4% (95%C1:308 to 488) of M1Cs were eqmvalent (within plusminus 1 log2), 60.6% (95%CI: 51.2 to 69.2) were major errors (> plusminus 1 log2) and 3% (95%CI: 0.8 to 10.4) very major errors (change in susceptibility pattern). MLxed infection were tound in 6 out of the 43 cases examined (13,9%). In 4 cases, MZR strains were l-log10 less numerous then MZS. hr the remaining 2 cases, the MZR strains were 2-3-1og10 less numerous, which caused the two misdiagnoses Conclusions: When compared to AD, the agreement of E-test (98.2%; 95%CI: 93.6 to 99.5), and DD (97.2%; 95%CI: 92.2 to 99.13 was high, Mixed infectiom have been shown to be a possible responsible of this small disagreement.

M2055 Mosaic Transfer of Penicillin Binding Protein 1A (pbp-lA) and High-Level ~Lactam Antibiotic Resistance in Helicobacter pylori Moo~Jun Back, Dong H Kwon, Da~nd Y, Graham Background: Amoxlcillin is one most commonly used ~dactam antibiotics for antbH, pyIori therapies. However, amoxicillin resistant clinical H. pylofi isolates have recently been reported with reduced.-efficacy of anti-H, pyloti therapies. Aim: To understand mechanisms of highlevel resistance to }dactam antibiotics. Methods: Genomic DNA from clinical H. pytori isolate IHd with highdevel amoxicillin renstance (MIC >32 mg,q.) was inu'oduced into susceptible H. pylori 26695 by natural transtorntation and resulted in H. pylori 26695 reskstant to highlevds of f~-lactam antibiotics (MIC 8 to >32 rug/L) To isolate the resistance determinants a lambda genomic library using genomic DNA from the resistant H. pylon IH-1 was constructed and individual library clones were introduced mto susceptible H. priori 26695 with selection on agar plate containing amoxiciIlin (4 rng/L). Results: 2 of 300 ckmes containing a gene encoding penicillin binding protein 1A (pbp-lk) converted anmxicillin susceptible to resistant H. py/oti. DNA sequence and deduced amino acid sequence analyses revealed that the DNA sequence divergence of the pbp-1A was 2,4% (48 of' 1980; 97.6% identity) between the susceptible H, pylori 26695 and the resistant H, pylori IH-1 Nucleotides 957 to 1980 (transpeptidase domain) of the resistant H. priori IH-1, which includes 36 nucleotide substitutions, was 100% switched in the transformed resistant H. pytori 26695 while nucleotides 1 to 956 (glycosyltransferase domain) were unchanged in the same resistant strain (called mosaic transfer). The transpeptidase domain of PBP-1A in the resistant H. ~yIori had i0 amino acid substitutinns (E406 to A, 5417 to T, M515 to I, D535 to N, 5543 to R, T556 to S, N562 to Y, K648 to Q, R649 to K, and R656 to P), A PCR-amplified DNA fragment comprising the 10 amino acid substitutions (1023-bps) from the resistant H, p)loT{ was able

AGA Abstracts

M2058 Multidrug Resistant of Heficobacter pylori in Thailand Varocha -.-. Mahachai, Pinit -.-, Kullavanijaya, Duangporn -.-. Thong-Ngam, Rathagorn -.-. Vilaichone Background and Aim: High HdRobacter pyIori(H pylofi) resistance to metronidazole has been reported in Thailand for more than seven years and is increasing worldwide. There was no previous ~vport of multidrug resistance of H. D'lori(MDR-Hp) in Thailand and t?w reports in the world literatures, This study was designed to demonstrate the prevalence of muhidmg resistant of H. pylori in our population. Method: All patients were seen at

A-406