Helicobacter pylori infection in hemodialysis patients: Susceptibility to amoxicillin and clarithromycin

Abstract

AIM: To evaluate susceptibility of Helicobacter pylori to amoxicillin and clarithromycin in end-stage renal disease (ESRD) patients and non-uremic controls.

METHODS: The subjects with dyspeptic complaints were 33 ESRD patients and 46 age- and sex-matched non-uremic controls who exhibited H pylori on antral biopsy specimens. The two groups were age and sex matched. The H pylori strains’ pattern of susceptibility to amoxicillin and clarithromycin was investigated with the agar dilution technique.

RESULTS: None of the H pylori strains from either group showed resistance to amoxicillin with the agar dilution method. Twelve (36.4%) of the ESRD group strains and 7 (15.2%) of the control group strains showed resistance to clarithromycin, and this difference was statistically significant (P<0.05).

CONCLUSION: Resistance to amoxicillin does not appear to be an important problem in H pylori-infected ESRD and non-uremic patients in our region. In contrast, the rates of resistance to clarithromycin are high, particularly in the ESRD population.

Key Words: Helicobacter pylori, Chronic renal failure, Antibiotic resistance, Clarithromycin, Amoxicillin

INTRODUCTION

Helicobacter pylori (H pylori) infection is the most common chronic bacterial infection in humans. Estimates indicate that approximately 60% of the world population is colonized with this agent. In addition to the direct effects of H pylori, complications related to infection with this bacterium are serious public health problems[1]. H pylori is a gram-negative, spiral-shaped bacillus that colonizes the antrum of the stomach causing type B chronic-active antral gastritis. Research has established that a number of diseases of the gastrointestinal system are linked with H pylori infection. Specifically, this condition is known to play an important role in the pathogenesis of peptic ulcer disease, and is also associated with gastric cancer[2,3].

The ideal treatment for H pylori infection is yet to be discovered. Triple-agent protocols involving a proton-pump inhibitor and two antibiotics are frequently used[4-6]. Most patients with H pylori infection respond well to these regimens; however, therapy may fail due to antibiotic resistance, poor compliance, or treatment-related factors (number/doses of combined medications, dosing frequency, and/or treatment duration). Antibiotic resistance is an important cause of H pylori treatment failure. In order to administer optimal treatment in a given region, it is critical to know the antibiotic susceptibility pattern for the strains that exist in the area[7-10].

The epidemiological data concerning H pylori infection in end-stage renal disease (ESRD) patients are insufficient[11]. Most studies of the epidemiological features of H pylori infection have revealed similar findings in ESRD and non-uremic patients[11]. The prevalence rates of H pylori infection and dyspeptic complaints in ESRD patients are high. Most individuals with ESRD exhibit immune system dysfunction, and these patients also tend to require frequent invasive procedures. These factors result in increased rates of infection, and this explains the very high frequencies of antibiotic use in this population.

We designed a controlled study to investigate possible differences in antibiotic resistance pattern for H pylori-infected ESRD patients and non-uremic controls at a major center in Ankara, Turkey.

MATERIALS AND METHODS

Subjects

Thirty-three ESRD patients who arrived at our gastroenterology clinic with dyspeptic complaints were enrolled in the study. Forty-six dyspeptic control patients with normal renal function and no other known health problems also participated. The ESRD group comprised of 17 (51.5%) females and 16 (48.5%) males. The mean age of these patients was 42±14 years, and the mean hemodialysis duration 4.1±2.8 years. The control group comprised of 26 (56.5%) females and 20 (43.5%) males of mean age 43±14 years (Table 1). The two groups were age and sex matched (P>0.05). Each of the 79 subjects was investigated with upper gastrointestinal endoscopy. The exclusion criteria were as follows: prior treatment for H pylori infection; use of any antibiotic, proton-pump inhibitor, H2-receptor antagonist or bismuth-containing drugs in the 30 d before the study; negative H pylori culture from an antral biopsy specimen; and positive H pylori culture but insufficient growth for antibiotic susceptibility testing.

Table 1 Demographic characteristics and frequencies of antibiotic resistance in the two study groups.

	Non-uremic patients n = 46	ESRD patients n = 33	P
Mean age (yr)	43±14	42±14	>0.05
Female/Male	26/20 (43.5%)	17/16 (48.5%)	>0.05
Hemodialysis duration (yr)		4.1±2.8
Clarithromycin resistance	15.2% (7/46)	36.4% (12/33)	<0.05
Amoxicillin resistance	0% (0/46)	0% (0/33)

The study was conducted according to the guidelines of the Declaration of Helsinki. The study protocol was approved by the Local Ethics Committee of Baskent University. Patients were enrolled in the study after getting their written informed consent.

Endoscopic procedure

All the upper gastrointestinal system endoscopy procedures were performed under appropriate sedation (lidocaine 10 mg/puff for pharyngeal anesthesia and intravenous midazolam 2.5-7.5 mg for premedication) using the same videoendoscope (Olympus GIF Q240). Two biopsy specimens were collected in each case, one from the pre-pyloric region and one from the incisura angularis. Tissues were placed in selective medium (Brucella broth culture containing 20% glycerol) for transfer to the laboratory.

Culture

Within 4 h of the endoscopy procedure, each biopsy specimen was placed in 0.5 mL sterile saline and homogenized in a tissue homogenizer. Two droplets of each homogenate were placed in 7% sheep blood Brucella agar, and the plates were incubated in microaerophilic conditions at 37 °C for 3-7 d. Another sample was gram stained and examined under the microscope for gram-negative spiral bacilli. Isolates from the cultures were identified as H pylori based on typical colony morphology on selective agar, and with gram staining and oxidase, catalase and urease tests.

The bacteria cultured from each case were kept in separate cryo vials with 0.75 mL of 20% glycerol containing Brucella broth medium at -80 °C until susceptibility testing was done.

Minimum inhibitor concentration study

Each stock cultured specimen was warmed, re-plated on 7% sheep blood Brucella agar medium, and incubated in microaerophilic conditions at 37 °C for 3-5 d. The pure cultured H pylori strains were then tested for susceptibility to amoxicillin and clarithromycin using the agar dilution method, in accordance with the National Committee for Clinical Laboratory Standards (NCCLS)[12-14]. Using appropriate solvents, two-fold serial dilutions of each antibiotic were prepared resulting in final concentration ranges of 0.015-4.0 µg/mL for amoxicillin (Sigma Chemical Co.) and 0.015-8.0 µg/mL for clarithromycin (Abbott Laboratories). Five percent sheep blood containing Mueller-Hinton agar was used for the agar dilution method. Multiple suspensions of each H pylori strain to be tested were prepared in sterile saline, and were arranged in order of clarity according to the MacFarland 2 standard technique. Then a 3-µL droplet of each suspension was placed in each of the above-described antibiotic dilutions and on control plates. All strains were studied in pairs, and the H pylori strain NCTC 11637 was used as a control. The inoculated dilution tubes and plates were incubated in microaerophilic conditions at 37 °C for 3 d. For each isolate, the dilution tube with the lowest antibiotic concentration that showed no visible growth was recorded as the minimum inhibitory concentration (MIC). To determine whether or not a strain was susceptible to each antibiotic, we compared the recorded MIC values for amoxicillin and clarithromycin to previously reported MIC cut-off values for these drugs (0.5 and 1 µg/mL, respectively)[14-17].

Statistical analysis

Numeric values were expressed as mean±SD. The χ² test was used to compare mean values, and the Mann-Whitney U test was used to compare numeric values. P values <0.05 were considered to indicate statistical significance.

RESULTS

A total of 79 H pylori strains (one from each ESRD patient and control subject) were isolated from the antral biopsy specimens. The demographics for the two study groups are shown in Table 1. None of the H pylori strains from either group showed resistance to amoxicillin. Twelve (36.4%) of the 33 ESRD group strains and 7 (15.2%) of the 46 control group strains showed resistance to clarithromycin, and this difference was statistically significant (P = 0.03) (Table 1).

Table 2 shows the demographic characteristics of the ESRD patients and control subjects with clarithromycin-resistant and clarithromycin-susceptible H pylori strains. Of the twelve ESRD patients with H pylori strains that were resistant to clarithromycin, five were males and seven were females. The mean age of these patients was 42.9±12.6 years, and the mean age in the susceptible cases was 40.9±14.7 years. This age difference was not statistically significant (P>0.05). The mean hemodialysis durations in the subgroups of ESRD patients with clarithromycin-resistant and susceptible H pylori strains were 5.6±3.8 years and 3.3±1.8 years, respectively (P>0.05).

Table 2 Demographic characteristics of the ESRD and non-uremic patients with clarithromycin-resistant and susceptible H pylori strains.

	Resistant	Susceptible	P
ESRD patients
n	12	21
Mean age (yr)	42.9±12.6	40.9±14.7	>0.05
Female/Male	7/5	10/11
Hemodialysis duration (yr)	5.6±3.8	3.3±1.8	>0.05
Non-uremic patients
n	7	39
Mean age (yr)	49±17	42±14	>0.05
Female/Male	4/3	22/17

Of the seven non-uremic control patients with H pylori strains that were resistant to clarithromycin, three were males and four were females. The mean age of these patients was 49±17 years, and the mean age in the susceptible cases was 42±14 years. This age difference was not statistically significant (P>0.05).

DISCUSSION

H pylori infection is the most widespread bacterial infection in the world. Antibiotic resistance is important in the management of these patients, and knowledge of resistance pattern helps the clinician to establish the most appropriate treatment strategy[18-20].

The therapy for H pylori infection in ESRD patients and non-uremic patients are approximately the same[21]. Drugs that contain bismuth may be toxic for individuals with ESRD, and are generally avoided in this population[11]. The combination of proton-pump inhibitors, clarithromycin and amoxicillin is frequently used to treat H pylori infection in both uremic and non-uremic patients[4,20,22].

For some antibiotics, the in vivo and in vitro findings for H pylori susceptibility differ. Rates of antibiotic resistance in H pylori infection, particularly resistance to clarithromycin, are rising dramatically in Turkey and many other countries throughout the world, so it is more important than ever to be aware of this issue[23,24].

One important barrier in the approach to this problem is the lack of a standard system for assessing H pylori microorganism susceptibility to antibiotics. The NCCLS subcommittee recommends the agar dilution method for this purpose, and this was our basis for using this technique in our study[14]. The NCCLS MIC value for H pylori resistance to clarithromycin is 1 µg/mL. There is no standard MIC value for H pylori resistance to amoxicillin, so we used the lowest value in the literature (0.5 µg/mL) as the cut-off in our study[14-17].

The in vitro efficacy of amoxicillin for H pylori infection is high; however, when amoxicillin is used as a single therapeutic agent, this rate drops to approximately 20% due to low efficacy at acid pH[25]. This is the reason why amoxicillin is used in combination with anti-secretory drugs[23]. A recent report has identified a number of H pylori strains that are resistant to amoxicillin[26-29], but none of the isolates in our study showed resistance to this agent.

Clarithromycin is the antibiotic to which H pylori is most sensitive in vitro, and this agent is widely used in combination regimens. Oral administration yields high serum and tissue concentrations, and the drug is stable in acid environments. Some authors have identified resistance to clarithromycin as the most important problem in H pylori treatment failure[30,31]. It has been reported that clarithromycin resistance results in significantly lower H pylori eradication rates[7], and studies from various nations have documented increasing rates of H pylori resistance to this agent in recent years[32-34]. Rates of H pylori resistance to clarithromycin are higher in countries where macrolide antibiotics are frequently used[32]. The estimated range of resistance to this antibiotic in H pylori-infected patients in various nations and regions of the world is 1-15%[32,35,36].

In our H pylori-infected study subjects, the rates of H pylori resistance to clarithromycin were 15.2% in the otherwise healthy controls and 36.4% in the ESRD patients. These figures are both very high, and provide further evidence that H pylori resistance to clarithromycin is on the rise in our country. If this problem continues to escalate, there will be even more serious problems treating H pylori infection in future. In addition to the high frequencies we observed in both study groups, the significantly higher rate of clarithromycin resistance in our ESRD group compared to the controls is of particular concern. It is important to be aware that the risk of H pylori resistance to this agent is likely to be higher in a patient who takes antibiotics for a variety of reasons. It is essential to know the antibiotic susceptibility of H pylori strains in order to identify what drug combination will be effective and how to prevent antibiotic resistance, to avoid using inappropriate drugs, and to decrease costs. To our knowledge, this is the first study that has examined H pylori resistance to amoxicillin and clarithromycin in H pylori-infected ESRD patients.

In conclusion, it appears that H pylori resistance to amoxicillin is not a major problem in ESRD and non-uremic patients in Turkey. However, the H pylori isolates from the ESRD patients in this series showed high rates of clarithromycin resistance, and expansion of this problem in all patient groups is a concern worldwide. Patients with ESRD have a high probability of H pylori resistance to clarithromycin due to immune compromise and extensive use of antibiotics. In order to effectively treat H pylori in this patient group, it is important to know the antibiotic susceptibility pattern of the H pylori microorganism present in each individual.