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Journal of the New Zealand Medical Association, 20-August-2004, Vol 117 No 1200

Antibiotic resistance in Helicobacter pylori: is it a problem in New Zealand?

Dilruba Ahmed, Heather Brooks, Michelle McConnell, Gil Barbezat

Abstract

Aims Infection with Helicobacter pylori requires antibiotic treatment when associated with upper gastrointestinal symptoms. Antibiotic susceptibility tests on this pathogen are rarely carried out and there is little information available on the incidence of antibiotic resistant strains in New Zealand. The aim of this study was to assess the susceptibility of H. pylori (cultured from gastric biopsies in Dunedin) to antibiotics that are commonly used in treatment.

Methods Over a 13-month period, gastric biopsies were obtained from selected patients undergoing endoscopy at Dunedin Public Hospital because of upper gastrointestinal symptoms. Biopsies were cultured for H. pylori and examined histologically. Fifty isolates from 50 patients with histological findings compatible with H. pylori infection were tested for sensitivity to metronidazole, clarithromycin, amoxycillin, and tetracycline using the epsilometer (E-) test.

Results Metronidazole resistance was detected in 10 (20%) isolates. These isolates were highly resistant to metronidazole and were not inhibited by 250mg/L. Resistance to clarithromycin, amoxycillin, and doxycycline was not detected.

Conclusions Clinicians should be aware that resistance to metronidazole appears to be common in H. pylori in New Zealand and that treatment regimens including this antibiotic may be less effective as a result. Although clarithromycin resistance was not detected, it is becoming increasingly problematical overseas and has been recorded in Auckland.

The association of the Gram negative, spiral bacterium Helicobacter pylori with gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma is now well established.1

Helicobacter pylori has a worldwide distribution, and serological studies in Auckland, New Zealand indicate infection is particularly common among Maori and Pacific Island communities.2 Prevalence is much lower amongst the European population, particularly in the South Island.3,4 Successful eradication of H. pylori infection depends on a number factors, one of the most important being the use of an antibiotic regimen to which the infecting strain is susceptible.5 Triple therapy combining an acid inhibiting agent (proton pump inhibitor) with clarithromycin and either amoxycillin or metronidazole is recommended as a first-line treatment in the Maastricht Consensus Report .

Second-line therapy should include a combination of an acid inhibitor with bismuth salt, metronidazole, and tetracycline or quadruple therapy (standard triple therapy as above plus bismuth).6,7 In recent years, the emergence of antibiotic-resistant strains has been widely reported, and these pose a significant threat to the successful treatment of H. pylori infection.5,6,8–10 For most bacterial infections, antibiotic susceptibility testing is routinely carried out but the specialised growth requirements of H. pylori preclude such tests in most diagnostic medical laboratories. There is a dearth of published New Zealand studies which include data on the antibiotic susceptibility of this pathogen.11,12 In practice, H. pylori infections are currently being treated in most centres without any up-to-date knowledge concerning the incidence of resistant strains.

The aim of the present study was to determine the antibiotic susceptibility of H. pylori isolated from gastric biopsies of patients referred to Dunedin Public Hospital because of upper gastrointestinal symptoms.

Materials and methods

Patients—Over a 13-month period, patients presenting for routine gastroscopy in the Endoscopy Unit of Dunedin Public Hospital were invited to participate in the study. The patient inclusion criteria comprised a history of upper gastrointestinal tract complaints requiring histological definition, symptoms suggestive of peptic ulcer, and where gastric biopsies were indicated from the physical findings at endoscopy.

Patients who did not give consent, or who had medical contraindications for taking biopsies, were excluded from the study. Ethical approval for the collection of extra biopsies was granted by the Otago Ethics Committee, Health Funding Authority, Dunedin.

Gastric biopsy samples—Upper endoscopy was performed and duplicate gastric biopsy samples from both the antrum and the corpus of each patient were obtained. One pair of samples (antrum and corpus) was subjected to histopathological examination in the Histopathology Department of Dunedin Public Hospital, and Giemsa-stained H. pylori identified by light microscopy. The other pair of samples was cultured for H. pylori as described below.

Samples for bacteriological examination were placed in Stuart’s Transport medium (Oxoid, Hampshire, England), transported to the laboratory on ice and processed within 2 hours. Cleaning and sterilisation of endoscopes and biopsy equipment were maintained according to nationally agreed criteria and a monthly sterility check performed on the endoscopic flush fluid.13

Culture and identification of H. pylori—Biopsy samples were cultured on Columbia agar with Dent H. pylori antibiotic supplement (Oxoid, Hampshire, England), containing 7% laked horse blood (Invitrogen, Auckland, New Zealand) and incubated in a microaerophilic atmosphere for up to 10 days as described by Goodwin.14 Colonies were identified as H. pylori as previously described.14 H. pylori NCTC 11637 (type strain) was used as a positive control for the culture conditions and identification tests.

Antibiotic susceptibility tests—Antibiotic susceptibility was determined on Mueller Hinton agar (Difco, Becton Dickinson & Co., Sparks, Maryland, USA ) containing 5% sheep blood (Invitrogen, Auckland, New Zealand) by the epsilometer test using metronidazole, clarithromycin, amoxycillin, and doxycycline E-test strips (AB Biodisk, Solna, Sweden). The tests were carried out according to the manufacturer’s instructions. Strains were considered resistant when the MIC for metronidazole was >8 mg/L; clarithromycin >1 mg/L; amoxycillin >1mg/L; and doxycycline >1 mg/L.15

H. pylori NCTC 11637 was used as a sensitive control.

Results

H. pylori was cultured from the biopsies of 58 of the 134 patients recruited for the study. Histopathological examination revealed H. pylori along the mucosal surface in 57 of these patients and sections of the gastric mucosa showed typical infiltration of mixed inflammatory cells composed of lymphocytes, plasma cells, and occasional polymorphs and eosinophils. A single patient was culture positive, but histology negative and endoscopic examination in this patient indicated the presence of gastritis, duodenitis and duodenal ulcer consistent with H. pylori infection.

In the 58 H. pylori culture positive patients, the clinical findings at endoscopy were: 22 patients had duodenal ulcers; 10 had gastric ulcers; 4 had duodenitis; and 12 had gastritis. In the remaining 10 patients, findings were inconclusive. There was no evidence of malignancy in any of the patients.

Fifty H. pylori isolates from 50 patients were available for E-tests (the remaining 8 isolates could not be satisfactorily subcultured) and the results are shown in Table 1. Resistance to clarithromycin, amoxycillin, and doxycycline was not detected—but isolates from 10 (20%) of the patients were resistant to metronidazole (95% confidence interval: 8.9–31.1%).

Table 1. Susceptibility to antibiotics of H. pylori cultured from gastric biopsy samples of patients attending the Endoscopy Unit of Dunedin Public Hospital

Antibiotic	Resistant isolates No. (%)	Range of MICs values in mg/L
		Resistant isolates	Sensitive isolates
Metronidazole Clarithromycin Amoxycillin Doxycycline	10 (20) 0 0 0	>256 ND ND ND	<0.016 <0.016 <0.016 0.19–1

ND=not detected

For the sensitive isolates, the minimum inhibitory concentrations (MICs) of clarithromycin, amoxycillin, and metronidazole were well below the break points for resistance. However, the metronidazole resistant strains were extremely resistant having MICs of >256mg/L.

Discussion

The most significant finding of this study was the detection of H. pylori that were highly resistant to metronidazole in 20% of the patients. A standardised method for assessing the susceptibility of H. pylori to metronidazole has not been established because this antibiotic is a pro-drug and reduction to the active form by the bacteria is influenced by the conditions of the test.9 Nevertheless, the high degree of resistance suggests that the efficacy of a regimen in which metronidazole featured would be compromised.

Using triple therapy including metronidazole, Megraud et al found the eradication rate of H. pylori infection was 96% when the bacteria were sensitive to the antibiotic but only 45% when they were resistant.16 Fraser et al found a similar decrease in the eradication rate when the infecting strain was metronidazole-resistant but noted that when clarithromycin was included in the therapeutic regimen, metronidazole resistance had a much smaller impact.12

The proportion of isolates resistant to metronidazole (20%) was somewhat lower in the present study compared to other parts of the world. Metronidazole resistance rates have been found to vary geographically with very high rates (80-90%) being recorded in China, Zaire, and Bangladesh.5

In a study carried out in Auckland, Fraser et al noted that 32% of H. pylori isolates were resistant to metronidazole, and Mollison suggests the incidence is similar in Australia.11,17 However, a multi-centre study, carried out by Katelaris et al, on H. pylori from Australia and New Zealand recorded a much higher incidence of primary metronidazole resistance (56%,) but failed to distinguish between isolates from each country.18

Factors contributing to the prevalence of metronidazole-resistance probably include the widespread use of nitro-imidazoles to treat gynaecological and Giardia infections or previous treatment of H. pylori infection with this drug, especially if the patient failed to take the whole course.19 Antibiotic exposure exerts a selective pressure on resistant mutants within the sensitive H. pylori population colonising the stomach;20 these become predominant as the sensitive bacteria are eradicated. Giardiasis is prevalent in many parts of New Zealand21 and, as long as metronidazole remains the treatment of choice, it appears unlikely that resistance in H. pylori to this drug will decrease over time. However, this statement must be qualified in view of the recent downward trend in metronidazole MICs in Australian isolates despite the overall consumption of this antibiotic remaining the same.15

In the present study, all isolates were sensitive to amoxycillin and tetracycline and, on a worldwide basis, H. pylori resistance to these antibiotics seem to be infrequent.8,17 Clarithromycin resistance was also not detected in our study but the sample size was insufficiently large to exclude it altogether. Clarithromycin is particularly efficient in eradicating H. pylori from the stomach as it achieves high concentration in the gastric mucosa and becomes highly concentrated intracellularly.19 It is of some concern that clarithromycin resistance is being increasingly reported with rates in other countries ranging from 10-40% and, in an Auckland based study, Fraser et al recorded a resistance rate of 6.8%.12,17

There is no doubt that primary and acquired resistance to clarithromycin jeopardises the success of the first-line recommended treatment regimen. Alarcon et al noted the cure rate varied from 20–50% when laboratory tests indicated the H. pylori were clarithromycin resistant but 64–98% when they were susceptible.5 Grove and Koutsouridis detected a step-wise annual increase in the percentage of isolates resistant to clarithromycin over a 5-year period in Australia, and attributed this to a change in prescribing habits, notably the switch from erythromycin usage to roxithromycin and clarithromycin.15 Cross-resistance has been demonstrated with macrolide antibiotics and some H. pylori resistance may reflect the use of this class of antibiotics in the community.22

Conclusion

If non-compliance can be ruled out as a reason for the failure of treatment regimens (including metronidazole) to effect a cure for H. pylori infection, then antibiotic resistance must be considered.23 For first-line treatments (including clarithromycin and amoxycillin), treatment failure is less likely to be due to antibiotic resistance. Metronidazole resistance in H. pylori is a problem in New Zealand, and is likely to continue to be so in the foreseeable future. The possibility of there being regional variations in the antibiotic susceptibility of H. pylori cannot be ruled out, and any future studies should take this into account. Further advances in molecular techniques may enable antibiotic resistance to be detected without the need to culture the bacteria, thus facilitating more successful treatment of infection caused by this very common pathogen.

Author information: Dilruba Ahmed, Postgraduate Student, Department of Microbiology, Dunedin School of Medicine, University of Otago, Dunedin; Heather Brooks, Visiting Scientist, Department of Microbiology, Dunedin School of Medicine, University of Otago, Dunedin; Michelle McConnell, Lecturer, Department of Microbiology, Otago School of Medical Sciences, University of Otago, Dunedin; Gil Barbezat, ‘Mary Glendining Professor of Medicine’, Department of Medicine, Dunedin School of Medicine; University of Otago, Dunedin

Acknowledgements: This study was supported by a University of Otago Bequest Fund grant. The assistance of the nursing staff of the Gastroenterology Endoscopy Unit, Dunedin Public Hospital is also gratefully acknowledged.

Correspondence: Dr Michelle McConnell, Department of Microbiology, Otago School of Medical Sciences, University of Otago, PO Box 56, Dunedin. Fax: (03) 479 8540; email: michelle.mcconnell@stonebow.otago.ac.nz

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