Resistance to Antibiotics and Characterization of Helicobacter pylori Strains Isolated from Antrum and Body from Adults in Mexico

Abstract

Background and Aims: Resistance to antibiotics is common in patients with Helicobacter pylori infection in more than one anatomic stomach site. We assessed whether the antibiotic resistance varies according to virulence factors of the bacteria as well as to the age and gender of individuals infected in two anatomic sites. Methods: H. pylori strains were isolated from an antral and corpus biopsy from 90 patients with gastric ambulatory who had not received any previous therapy. Susceptibility to metronidazole and clarithromycin was assessed by E-test, and vacA and cagA genotypes were determined by polymerase chain reaction. Results: Dual resistance to metronidazole and clarithromycin was 3.3% in antrum and 4.4% in the corpus; heteroresistance was 19% and 5.5% for metronidazole and clarithromycin, respectively. Clarithromycin resistance significantly increased with age. Women showed a twofold increased risk for metronidazole-resistant strains in antrum (odds ratio = 2.85, 95% confidence interval 1.09 to 7.42). Virulence factors were not associated with antimicrobial resistance. Conclusion: Prevalence of resistance to clarithromycin may be increasing in this country. Antimicrobial susceptibility tests from different biopsy sites deserve attention.

Introduction

H elicobacter pylori is implicated in the etiology of gastric diseases ranging from superficial gastritis to gastric cancer, and it has been classified as a group I carcinogen.²⁸ In developing countries such as Mexico, more than 80% of adults are colonized with H. pylori, and there is no difference between urban and rural populations.³⁰ Colonization generally persists throughout life, except in those people effectively treated with antibiotics or in those who develop atrophic gastritis, usually late in life.^14,32 Eradication of H. pylori prevents peptic ulcer²⁵ and gastric adenocarcinoma,⁶ promotes the healing of early low-grade mucosa-associated lymphoid tissue (MALT) lymphoma,⁵ and possibly provides relief for nonulcer dyspepsia.^17,24 In general, the guidelines for treating patients in whom H. pylori infections were diagnosed consisted of traditional clarithromycin-containing triple therapy in combination with a proton pump inhibitor (PPI). However, recently results regarding H. pylori eradication treatments follow this regimen point to the fact that they do not reliably offer a ≥80% cure rate, due mainly to the increased prevalence of antibiotic resistance. Recently, alternative treatments have been proposed.¹⁵ These new treatments usually consist of a PPI in combination with two or three antibiotics, among which amoxicillin, clarithromycin, metronidazole, and tetracycline are included. The primary resistance rate to the two most commonly used antibiotics, clarithromycin and metronidazole, is an important factor to be considered.²⁷ Thus, the choice of drug regimen should be made based on the knowledge of recent data on the resistance rates to these antibiotics in a given geographical area.

Another factor that could affect the success of therapy is the fact that an individual might have a mixed H. pylori infection with differing antimicrobial susceptibilities in different parts of the stomach.^19,21,33 However, in our country, there is still very little information on the prevalence of antimicrobial-resistant H. pylori strains isolated from gastric biopsies from different anatomical sites of the stomach.

We assessed whether clarithromycin, metronidazole, and amoxicillin resistance vary according to the site of isolation and the bacterial virulence factors (vacA (s/m) alleles and the cagA status) as well as the age and gender of Mexican ambulatory patients infected with H. pylori.

Materials and Methods

Study design

A cross-sectional study was conducted during the period from January 2002 to December 2004. It included 460 ambulatory patients who were attending seven hospitals in Mexico City (Instituto Nacional de Cancerología, Hospital General Manuel Gea González, Hospital General de México, Hospital de Especialidades la Raza, Hospital de Especialidades del Centro Médico Nacional Siglo XXI, Hospital de Oncología del Centro Médico Nacional Siglo XXI, Hospital de Especialidades Ignacio Zaragoza).

Patients

Eligible patients were permanent residents of Mexico City who were at least 15 years old and who agreed to donate a sample of their stomach biopsy. This was obtained from a gastroscopy that they had undergone with diagnostic purposes, having had no anti H. pylori treatment (antibiotics, histamine-2 blockers, or PPIs) in the 15 days before the endoscopy. Four hundred and sixty agreed to participate in the study. Two biopsy specimens were obtained for culture of H. pylori, one from the antrum and the other from the corpus. Only 90 (19.5%) individuals were culture positive for H. pylori obtained from both sites. All patients provided written informed consent, and the study protocol was approved by the Ethics Review Board of the National Institute of Public Health.

Bacterial strains

Each biopsy specimen was homogenized using a tissue grinder and then inoculated onto tripticase soy agar plates (Becton Dickinson, Sparks, MD) supplemented with 10% sheep blood, and Dent antibiotic selective supplement (Oxoid, Basingstoke, United Kingdom). The plates were incubated in an air atmosphere with 10% v/v CO₂ at 37°C for 3 to 5 days until visible colonies appeared. The strains were identified by Gram staining, colony morphology, and biochemical tests (oxidase, catalase and urease positive). The isolates were stored at −70°C in 1.5 ml Brain Heart Infusion Broth (Becton Dickinson) with 30% glycerol, until they were used.

Susceptibility testing

Fresh H. pylori isolates from a 72 hour subculture, taken from a blood agar plate, were diluted in sterile Müeller-Hinton broth (Becton Dickinson) and adjusted to a 3.0 McFarland standard (≥10⁸cfu/ml of viable cells).

The bacterial suspension was spread on Petri plates (90 mm diameter) containing Müeller Hinton agar (Becton Dickinson) supplemented with 10% sheep blood and 10% fetal bovine serum (Gibco, BBL Bethesda, MD) under the same microaerophilic conditions just mentioned (5% O₂, 10% CO₂) at 37°C for 72 hours. An E-test strip was placed onto the agar when the surface of the plate (90 mm diameter) was dry. E-test strips of amoxicillin (concentration ranges: 0.016–256 μg/ml), clarithromycin (0.016–256 μg/ml), and metronidazole (0.06–256 μg/ml) were obtained from AB Biodisk (Solna, Sweden).

H. pylori strain ATCC 43504 was used as a quality control in all resistance determinations. H. pylori strains were considered resistant if their MIC was ≥8 μg/ml for metronidazole, ≥2 μg/ml for clarithromycin, and ≥4 μg/ml for amoxicillin.^8,20,31 Growth control plates without antibiotics were included in each series of tests.

Analysis of vacA and cagA genes

For polymerase chain reaction genomic typing, DNA was extracted from a pool of colonies obtained from each biopsy site. The colonies were harvested with sterile cotton swab applicators. Genomic DNA was isolated using the GenomicPrep Cells and Tissue DNA isolation kit (Amhersam Pharmacia Biotech, Piscataway, NJ) according to the manufacturer's recommendations.

The presence of cagA and vacA (s and m region) was determined by multiplex polymerase chain reactions as previously described.⁴ The primers used for the amplification of vacA s1/s2¹ and m1/m2¹⁸ alleles and for cagA⁴ were previously reported.

Statistical analysis

Antibiotic susceptibility to metronidazole or to clarithromycin by site of isolation and simultaneous resistance to clarithromycin and metronidazole in each site were compared with the chi-square test. Mean age differences, classified by gender, among all patients harboring susceptible and resistant strains, and divided according to isolation site, were obtained by Student's t-test.

The frequencies of cagA/vacA genotypes of H. pylori were estimated according to antibiotic susceptibility by site of isolation, comparing the observed and expected frequencies with the chi-squared test.

The association of age, gender, and virulence markers (cagA and vacA s/m) with antibiotic susceptibility, according to the site of isolation, was evaluated by multivariate logistic regression models. All statistical analyses used STATA version 9.2 (STATA Corporation, College Station, TX). A probability (p) value of less than 0.05 was considered significant.

Results

A total of 460 patients from seven hospitals in Mexico City were included in a prospective study. H. pylori was successfully cultured from the two gastric sites (antrum and corpus) in 90 patients (19.5%). E-test determinations and cagA and vacA s/m genotyping were conducted for each isolate.

Antibiotic susceptibility

All recovered H. pylori isolates were assayed against clarithromycin, amoxicillin, and metronidazole resistance. All the isolates turned out to be susceptible to amoxicillin.

Antibiotic susceptibility was examined to determine whether susceptibility patterns were the same among paired isolates from antrum and body. When analyzing metronidazole or clarithromycin susceptibility for antrum versus corpus, we found that most patients (90% [81/90]) had clarithromycin-susceptible strains in both sites. On the other hand, the prevalence of patients harboring resistant strains simultaneously in the antrum and body was higher for metronidazole than for clarithromycin (52% [47/90] vs. 4.4% [4/90], respectively). The prevalence of patients infected concurrently with mixed susceptible/resistant strains was 19% (17/90) for metronidazole and 5.5% (5/90) for clarithromycin. These differences were statistically significant (p < 0.05). The prevalence of clarithromycin resistance was slightly more frequent among isolates obtained from the body compared with those obtained from the antrum (8.9% [8/90] vs. 5.6% [5/90]) (Table 1A). The overall resistance to metronidazole of isolates obtained from the antrum was 61% and for the corpus was 62% (Table 1A). Among metronidazole-resistant strains, most had high MICs (>256 μg/ml), both in antrum and corpus. Clarithromycin was very active: 50% of the strains showed MICs of <0.016 μg/ml both in antrum and corpus sites (data not shown).

Table 1.

Proportion of Patients Infected with Antimicrobial-Resistant Helicobacter pylori According to the Site of Isolation, Antrum, and/or Corpus

(A)
Site		Antibiotic
Antrum	Corpus	Metronidazole susceptibility^a n (%)	Clarithromycin susceptibility^a n (%)
Susceptible	Susceptible	26 (29)	81 (90)
Susceptible	Resistant	9 (10)	4 (4.4)
Resistant	Susceptible	8 (9)	1 (1.1)
Resistant	Resistant	47 (52)	4 (4.4)
Total		90 (100)	90 (99.9)

(B)
Antibiotic		Site
Metronidazole	Clarithromycin	Antrum n (%)	Corpus n (%)
Susceptible	Susceptible	33 (36.7)	30 (33.3)
Susceptible	Resistant	2 (2.2)	4 (4.4)
Resistant	Susceptible	52 (57.8)	52 (57.8)
Resistant	Resistant	3 (3.3)	4 (4.4)
Total		90 (100)	90 (99.9)

p < 0.05 for Chi² test.

Simultaneous resistance to clarithromycin and metronidazole was also investigated for each site (Table 1B). Infection with metronidazole-resistant/clarithromycin-susceptible strains was the most frequent for both sites, 57.8% (52/90). The prevalence of patients harboring strains that were resistant to both antibiotics in the antrum and corpus was 3.3% (3/90) and 4.4% (4/90), respectively. The prevalence of patients infected with strains that were susceptible to both antibiotics was 36.7% (33/90) for the antrum and 33.3% (30/90) for the corpus. The prevalence of patients harboring clarithromycin-resistant/metronidazole-susceptible strains was 2.2% (2/90) for the antrum and 4.4% (4/90) for the corpus. These differences were not statistically significant (Table 1B).

Epidemiological data

The analyses of H. pylori antibiotic resistance in terms of the age and gender of the patients showed that the average age of individuals harboring strains resistant to metronidazole in the antrum was significantly higher (51 ± 13.9) than the average age of those harboring susceptible strains in the same site (43.8 ± 13.8) (p < 0.05). With respect to the corpus, no differences were observed in terms of metronidazole susceptibility (Table 2). However, in the case of clarithromycin, the average age of patients harboring resistant strains in both antrum and corpus was significantly higher than the average age of those harboring susceptible strains, with an average age of 63.8 ± 12.0 versus 47.3 ± 13.8 (p < 0.05) for the antrum and 59.25 ± 11.9 versus 47.16 ± 14.03 (p < 0.05) for the corpus (Table 2). Moreover, the average age of women harboring metronidazole-resistant strains in the antrum was higher than the average age of women harboring susceptible strains (52 ± 13.2. vs. 43.9 ± 13.9) (p < 0.05). On the other hand, resistance to clarithromycin was associated with older women in both antrum and corpus, with an average age of 63.8 ± 12.0 versus 47.6 ± 13.4 (p < 0.05) and 63.3 ± 11.0 versus 47.34 ± 13.4 (p < 0.05), respectively. The rates of H. pylori metronidazole- and clarithromycin-resistant strains were not associated with the age of male patients (Table 2).

Table 2.

Mean Age, Gender Differences Among Patients Harboring Susceptible and Resistant Strains, According to Site of Isolation

	Metronidazole				Clarithromycin
	Antrum (n = 90)		Corpus (n = 90)		Antrum (n = 90)		Corpus (n = 90)
	MZ^R	MZ^S	MZ^R	MZ^S	CH^R	CH^S	CH^R	CH^S
All	(n = 55)	(n = 35)	(n = 56)	(n = 34)	(n = 5)	(n = 85)	(n = 8)	(n = 82)
Mean age (year) ± SD	51.0 + 13.9^a	43.8 + 13.8	49.7 + 13.8	45.79 + 14.6	63.8 + 12.0^a	47.3 + 13.8	59.25 + 11.9^a	47.16 + 14.03
Female (n = 58)	(n = 36)	(n = 22)	(n = 41)	(n = 17)	(n = 5)	(n = 53)	(n = 6)	(n = 52)
Mean age (year) ± SD	52.0 + 13.2^a	43.9 + 13.9	50.14 + 14.0	46.2 + 13.8	63.8 + 12.0^a	47.6 + 13.4	63.3 + 11.0^a	47.34 + 13.4
Male (n = 32)	(n = 19)	(n = 13)	(n = 15)	(n = 17)	(n = 0)	(n = 32)	(n = 2)	(n = 30)
Mean age (year) ± SD	49.0 + 15.3	43.6 + 14.1	48.5 + 13.9	45.3 + 15.8	0	46.8 + 14.9	47.0 + 0.0	46.8 + 15.3

p < 0.05 for t-test.

MZ^R, metronidazole resistant; MZ^S, metronidazole susceptible; CH^R, clarithromycin resistant; CH^S, clarithromycin susceptible; SD, standard deviation.

The analyses of prevalence of vacA genotypes and cagA status in strains isolated from both gastric sites showed a strong association between the cagA⁺ and the vacA s1/m1 genotype, whereas the cagA negative strains were related to the s2m2 genotype in the case of isolates obtained from both sites, as it was previously reported.² There was no significant association between metronidazole and clarithromycin resistance and the cagA⁺ and s1/m1 genotypes.

The adjusted logistic regression multivariable model showed that patients have a higher risk of harboring metronidazole resistance in the antrum for each year of aging (odds ratio [OR] = 1.12, 95% confidence interval [CI] 1.01 to 1.23); a similar effect was observed for clarithromycin-resistant strains in both antrum and corpus (OR = 1.04, 95% CI 1.0 to 1.07, and OR = 1.06, 95% CI 1.0 to 1.12), respectively (Table 3). On the other hand, women have a higher risk of being infected with metronidazole-resistant strains in the corpus than men (OR = 2.85, 95% CI 1.09 to 7.42; Table 3). Virulence markers cagA and vacA s/m were not associated with either metronidazole or clarithromycin resistance in any of the investigated sites.

Table 3.

Multivariate Analysis by Logistic Regression Model, Evaluating Age, Gender, and Virulence Markers (cagA and vacA s/m) Status According to Antibiotic Susceptibility, by Site of Isolation

	Metronidazole		Clarithromycin
	Antrum OR (95% CI)	Corpus OR (95% CI)	Antrum OR (95% CI)	Corpus OR (95% CI)
Age (years)	1.04 (1.0–1.07)^a	1.02 (0.98–1.05)	1.12 (1.01–1.23)^a	1.06 (1.0–1.12)^a
Gender
Male	1.0	1.0	—	1.0
Female	1.03 (0.39–2.67)	2.85 (1.09–7.42)^a	—	1.55 (0.25–9.45)
vacA genotype
s2m2	1.0	1.0	1.0	1.0
s1m2/s2m1	0.55 (0.057–5.34)	4.62 (0.53–40.4)	—	1.61 (0.08–29.5)
s1m1	0.67 (0.14–3.17)	2.58 (0.59–11.3)	4.76 (0.2–109.7)	0.9 (0.09–8.88)
cagA genotype
Negative	1.0	1.0	1.0	1.0
Positive	0.69 (0.21–2.28)	1.0 (0.3–3.26)	0.25 (0.01–3.66)	0.79 (0.11–5.33)

p < 0.05.

OR, odds ratio; CI, confidence interval.

Discussion

In this study, the resistance to metronidazole and clarithromycin against H. pylori isolates obtained from the antrum and corpus of Mexican patients during a period between 2002 and 2004 was confirmed. Our results showed that the overall resistance rate to clarithromycin has increased in our country from 4% between 1997 and 2001⁷ to 6.98% between 2002 and 2004. However, previous studies have reported that clarithromycin resistance was as high as 25% between 1995 and 1997,³¹ and 12.9% in 2002.¹² These discrepancies were probably due to the kind of patients involved in each study: the subjects came from different hospitals, from diverse socioeconomic levels and even from different regions in the country. Therefore, the high prevalence of resistance to clarithromycin previously observed in our country might have been due to the clinical use of oral macrolides for the treatment of respiratory tract infections as well as for the treatment of mycoplasma, chlamydia, and ureaplasma infections. However, data on macrolide use are not available in our country. Even so, we believe it is important for clinicians to become aware of the tendency toward the increase of the prevalence of clarithromycin-resistant strains in our country. This is especially important if we take into account that the traditional first-line treatment consisting of clarithromycin-containing triple therapy in combination with a PPI achieves a cure rate ≤80%, which could be due to the increased prevalence of clarithromycin resistance that has been observed in other regimens containing this agent.^{3,9,10,11,22,23} On the other hand, we also found a high rate of resistance to metronidazole. Nevertheless, it has been reported that pretreatment antibiotic resistance to metronidazole does not always predict decreased clinical efficacy.^11,15,16

In agreement with other studies,^21,26 we found heteroresistance to metronidazole and clarithromycin when comparing pairs of isolates from antrum and corpus. We found that 19% of the patients harbored a mixed infection with metronidazole-susceptible/resistant strains, whereas 5.5% of the subjects were infected simultaneously with clarithromycin-susceptible/resistant strains. In this study, we found that the overall prevalence of clarithromycin resistance was slightly more frequent among isolates obtained from the corpus compared with those obtained from the antrum (8.8% vs. 5.5%). Similar results were reported by Masuda et al.,²³ who reported a higher prevalence of clarithromycin-resistant strains in the corpus than in the antrum (18.46 [12/65] vs. 13.8% [9/65]). The same authors suggested that clarithromycin-resistant strains survive clarithromycin exposure and become dominant in the stomach.

We found that older age was significantly associated with clarithromycin resistance, particularly among women in whom a high frequency of clarithromycin-resistant strains was found in both sites. Significantly high rates of metronidazole resistance in older women were also observed in isolates obtained solely from the antrum. Further, a multivariable analysis showed that women were potentially at a higher risk than men of harboring metronidazole-resistant strains in the corpus (OR = 2.85). This resistance to metronidazole could be explained, because most of the gynecological infections as well as some protozoan ones are likely to be treated with this drug.

In our study, 3.3% and 4.4% of the patients displayed an infection that was resistant to both metronidazole and clarithromycin in the antrum and corpus, respectively. This dual resistance would certainly affect the success of treatment; nevertheless, it has been recently reported that some treatment could overcome this problem.^13,34 Clarithromycin is one of the most frequently used antibiotics for the eradication of H. pylori in Mexico. Therefore, our results suggest that alternative drug regimens might be considered in the near future for the eradication of H. pylori in our community.

We also found a high prevalence of resistance to metronidazole among strains harboring the cagA⁺/vacA s1/m1 genotype both in the antrum and the body. Some studies have reported that virulent strains (cagA⁺/vacA s1/m1) could be more easily eliminated than less virulent strains (cagA⁻/vacA s2/m2).²⁹ Thus, in patients carrying cagA⁺/s1/m1 metronidazole- and/or clarithromycin-resistant strains, it would be more difficult to eliminate H. pylori and they might be at a higher risk of developing severe gastroduodenal diseases such as peptic ulcer and gastric cancer.

In conclusion, prevalence of resistance to clarithromycin may be increasing in this country. We found the rate of clarithromycin resistance was significantly associated with older age, particularly in women. According to previous reports, patients can be colonized with mixed metronidazole and clarithromycin susceptible/resistant strains at the same time, in the same or different sites. Therefore, the present results indicate the need to test the antimicrobial susceptibility of bacteria isolated from both gastric sites (antrum and corpus) of the same patient in order to diagnose clarithromycin-resistant H. pylori infection correctly.

Study Group

The following hospitals participated (names of enrolled subjects):

Hospital General Dr. Manuel GEA González: Dr. Mucio Moreno Portilla, Jefe de Cirugía Endoscópica. Dr. Gonzalo Rodríguez, Jefe de Gastroenterología. Hospital de Especialidades CMN “La Raza”: Dra. Ana María Gómez, Jefe del Departamento de Patología; Dr. Enrique Paredes Cruz, Jefe del Departamento de Gastroenterología. Hospital General de México de la SSA: Dra. Rosario Valdez, Jefa del Servicio de Endoscopia de la Unidad de Gastroenterología; Dr. Bernal Sahagún, Jefe del Servicio de Gastroenterología. Hospital de Especialidades Centro Médico Nacional Siglo XXI: Dra. Margarita Dehesa Violante, Jefa del Servicio de Gastroenterología; Dra. Nayeli Ortiz, Jefe de Endoscopía. Hospital Regional “General Ignacio Zaragoza”: Dr. Rolando Armienta Sarabia, Jefe del Departamento de Endoscopia. Dr. Baldomero Hernández, Jefe de Patología. Instituto Nacional de Cancerología: Dr. Alejandro Mohar Betancourt, Director de Investigación; Dra. Angélica Hernández, Jefe de Endoscopía. Hospital de Oncología: Dra. Isabel Alvarado, Jefe de Patología; Dr. Marco Gutiérrez, Investigador responsible.

Footnotes

Acknowledgment

This work was financially supported by a grant of CONACyT (37195-M) from Mexico.

Disclosure Statement

No competing financial interests exist.

References

Atherton

J.C.

, Cao

, Peek

R.M.

Jr. , Tummuru

M.K.

, Blaser

M.J.

, Cover

T.L.

1995. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori. Association of specific vacA types with cytotoxin production and peptic ulceration. J. Biol. Chem., 270:17771–17777.

Atherton

J.C.

, Cover

T.L.

, Twells

R.J.

, Morales

M.R.

, Hawkey

C.J.

, Blaser

M.J.

1999. Simple and accurate PCR-based system for typing vacuolating cytotoxin alleles of Helicobacter pylori. J. Clin. Microbiol., 37:2979–2982.

Bago

, Halle

Z.B.

, Strinic

, Kucisec

, Jandric

, Bevanda

, Tomic

, Bilic

2002. The impact of primary antibiotic resistance on the efficacy of ranitidine bismuth citrate- vs. omeprazole-based one-week triple therapies in H. pylori eradication—a randomised controlled trial. Wien Klin Wochenschr., 114:448–453.

Chattopadhyay

, Patra

, Ramamurthy

, Chowdhury

, Santra

, Dhali

G.K.

, Bhattacharya

S.K.

, Berg

D.E.

, Nair

G.B.

, Mukhopadhyay

A.K.

2004. Multiplex PCR assay for rapid detection and genotyping of Helicobacter pylori directly from biopsy specimens. J. Clin. Microbiol., 42:2821–2824.

Chen

L.T.

, Lin

J.T.

, Tai

J.J.

, Chen

G.H.

, Yeh

H.Z.

, Yang

S.S.

, Wang

H.P.

, Kuo

S.H.

, Sheu

B.S.

, Jan

C.M.

, Wang

W.M.

, Wang

T.E.

, Wu

C.W.

, Chen

C.L.

, Su

I.J.

, Whang-Peng

, Cheng

A.L.

2005. Long-term results of anti-Helicobacter pylori therapy in early-stage gastric high-grade transformed MALT lymphoma. J. Natl. Cancer Inst., 97:1345–1353.

Cheung

T.K.

, Wong

B.C.

2008. Treatment of Helicobacter pylori and prevention of gastric cancer. J. Dig. Dis., 9:8–13.

Chihu

, Ayala

, Mohar

, Hernandez

, Herrera-Goepfert

, Fierros

, Gonzalez-Marquez

, Silva

2005. Antimicrobial resistance and characterization of Helicobacter pylori strains isolated from Mexican adults with clinical outcome. J. Chemother., 17:270–276.

DeLoney

C.R.

, Schiller

N.L.

2000. Characterization of an In vitro-selected amoxicillin-resistant strain of Helicobacter pylori. Antimicrob. Agents Chemother., 44:3368–3373.

Demir

, Gokturk

H.S.

, Ozturk

N.A.

, Arslan

, Serin

, Yilmaz

2009. Clarithromycin resistance and efficacy of clarithromycin-containing triple eradication therapy for Helicobacter pylori infection in type 2 diabetes mellitus patients. South Med. J., 102:1116–1120.

10.

Ducons

J.A.

, Santolaria

, Guirao

, Ferrero

, Montoro

, Gomollon

1999. Impact of clarithromycin resistance on the effectiveness of a regimen for Helicobacter pylori: a prospective study of 1-week lansoprazole, amoxycillin and clarithromycin in active peptic ulcer. Aliment. Pharmacol. Ther., 13:775–780.

11.

Fischbach

, Evans

E.L.

2007. Meta-analysis: the effect of antibiotic resistance status on the efficacy of triple and quadruple first-line therapies for Helicobacter pylori. Aliment. Pharmacol. Ther., 26:343–357.

12.

Garza-Gonzalez

, Perez-Perez

G.I.

, Alanis-Aguilar

, Tijerina-Menchaca

, Maldonado-Garza

H.J.

, Bosques-Padilla

F.J.

2002. Antibiotic susceptibility patterns of Helicobacter pylori strains isolated from northeastern Mexico. J. Chemother., 14:342–345.

13.

Graham

D.Y.

, Shiotani

2008. New concepts of resistance in the treatment of Helicobacter pylori infections. Nat. Clin. Pract. Gastroenterol. Hepatol., 5:321–331.

14.

Graham

D.Y.

, Lew

G.M.

, Klein

P.D.

, Evans

D.G.

, Evans

D.J.

Jr. , Saeed

Z.A.

, Malaty

H.M.

1992. Effect of treatment of Helicobacter pylori infection on the long-term recurrence of gastric or duodenal ulcer. A randomized, controlled study. Ann. Intern. Med., 116:705–708.

15.

Graham

D.Y.

, Fischbach

2010. Helicobacter pylori treatment in the era of increasing antibiotic resistance. Gut, 59:1143–1153.

16.

Graham

D.Y.

, de Boer

W.A.

, Tytgat

G.N.

1996. Choosing the best anti-Helicobacter pylori therapy: effect of antimicrobial resistance. Am. J. Gastroenterol., 91:1072–1076.

17.

Gwee

K.A.

, Teng

, Wong

R.K.

, Ho

K.Y.

, Sutedja

D.S.

, Yeoh

K.G.

2009. The response of Asian patients with functional dyspepsia to eradication of Helicobacter pylori infection. Eur. J. Gastroenterol. Hepatol., 21:417–424.

18.

Hunt

R.H.

, Fallone

C.A.

, Thomson

A.B.

1999. Canadian Helicobacter pylori Consensus Conference update: infections in adults. Canadian Helicobacter Study Group. Can. J. Gastroenterol., 13:213–217.

19.

Ikezawa

, Kashimura

, Kojima

, Aikawa

, Nakahara

, Mutoh

, Tanaka

1999. Pretreatment antimicrobial susceptibilities of paired gastric Helicobacter pylori isolates: antrum versus corpus. Helicobacter, 4:218–221.

20.

Iovene

M.R.

, Romano

, Pilloni

A.P.

, Giordano

, Montella

, Caliendo

, Tufano

M.A.

1999. Prevalence of antimicrobial resistance in eighty clinical isolates of Helicobacter pylori. Chemotherapy, 45:8–14.

21.

Kim

J.J.

, Kim

J.G.

, Kwon

D.H.

2003. Mixed-infection of antibiotic susceptible and resistant Helicobacter pylori isolates in a single patient and underestimation of antimicrobial susceptibility testing. Helicobacter, 8:202–206.

22.

Mahachai

, Thong-Ngam

, Noophun

, Tumwasorn

, Kullavanijaya

2006. Efficacy of clarithromycin-based triple therapy for treating Helicobacter pylori in Thai non-ulcer dyspeptic patients with clarithromycin-resistant strains. J. Med. Assoc. Thai, 89,Suppl 3:S74–S78.

23.

Masuda

, Hiyama

, Yoshihara

, Tanaka

, Shimamoto

, Haruma

, Chayama

2003. Necessity of multiple gastric biopsies from different sites for detection of clarithromycin-resistant Helicobacter pylori strains. Scand. J. Gastroenterol., 38:942–946.

24.

Ong

S.P.

, Duggan

2004. Eradication of Helicobacter pylori in clinical situations. Clin. Exp. Med., 4:30–38.

25.

Pimanov

S.I.

, Makarenko

E.V.

, Voropaeva

A.V.

, Matveenko

M.E.

, Voropaev

E.V.

2008. Helicobacter pylori eradication improves gastric histology and decreases serum gastrin, pepsinogen I and pepsinogen II levels in patients with duodenal ulcer. J. Gastroenterol. Hepatol., 23:1666–1671.

26.

Rimbara

, Noguchi

, Tanabe

, Kawai

, Matsumoto

, Sasatsu

2005. Susceptibilities to clarithromycin, amoxycillin and metronidazole of Helicobacter pylori isolates from the antrum and corpus in Tokyo, Japan, 1995–2001. Clin. Microbiol. Infect., 11:307–311.

27.

Romano

, Iovene

M.R.

, Russo

M.I.

, Rocco

, Salerno

, Cozzolino

, Pilloni

A.P.

, Tufano

M.A.

, Vaira

, Nardone

2008. Failure of first-line eradication treatment significantly increases prevalence of antimicrobial-resistant Helicobacter pylori clinical isolates. J. Clin. Pathol., 61:1112–1125.

28.

Schistosomes, liver flukes, Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Lyon, 7–14 June 1994. IARC Monogr. Eval. Carcinog. Risks. Hum, 1994; 61:1–241.

29.

Sugimoto

, Yamaoka

2009. Virulence factor genotypes of Helicobacter pylori affect cure rates of eradication therapy. Arch. Immunol. Ther. Exp. (Warsz)., 57:45–56.

30.

Torres

, Lopez

, Lazcano

, Camorlinga

, Flores

, Munoz

2005. Trends in Helicobacter pylori infection and gastric cancer in Mexico. Cancer Epidemiol. Biomarkers Prev., 14:1874–1877.

31.

Torres

, Camorlinga-Ponce

, Perez-Perez

, Madrazo-De la Garza

, Dehesa

, Gonzalez-Valencia

, Munoz

2001. Increasing multidrug resistance in Helicobacter pylori strains isolated from children and adults in Mexico. J. Clin. Microbiol., 39:2677–2680.

32.

Valle

, Kekki

, Sipponen

, Ihamaki

, Siurala

1996. Long-term course and consequences of Helicobacter pylori gastritis. Results of a 32-year follow-up study. Scand. J. Gastroenterol., 31:546–550.

33.

Wong

B.C.

, Wang

W.H.

, Berg

D.E.

, Fung

F.M.

, Wong

K.W.

, Wong

W.M.

, Lai

K.C

, Cho

C.H.

, Hui

W.M.

, Lam

S.K.

2001. High prevalence of mixed infections by Helicobacter pylori in Hong Kong: metronidazole sensitivity and overall genotype. Aliment. Pharmacol. Ther., 15:493–503.

34.

D.C.

, Hsu

P.I.

, Wu

J.Y.

, Opekun

A.R.

, Kuo

C.H.

, Wu

I.C.

, Wang

S.S.

, Chen

, Hung

W.C.

, Graham

D.Y.

2010. Sequential and concomitant therapy with four drugs is equally effective for eradication of H. pylori infection. Clin. Gastroenterol. Hepatol., 8:36–41.e1.