Abstract: Successful triple-therapy treatment for Helicobacter pylori infection depends upon metronidazole (Mz) susceptibility, and many hospital laboratories routinely screen H. pylori isolates for Mz resistance using disc diffusion methods. We report the importance of culture medium when testing for metronidazole susceptibility. In this laboratory, Mz resistance in strains of H. pylori from patients in our area was found in approximately 80%. In other areas, Mz resistance is found in approximately 30%. This high rate of Mz resistance was not reflected clinically. Added haemin (X factor) and menadione in the culture medium drastically reduced zone size to Mz and also interfered with the minimal inhibitory concentration (MIC) as determined by Etestt. When strains of H. pylori were re-tested on media which did not contain X factor or menadione, Mz resistance fell from 80% to 39%, a level similar to that seen in other areas.
Key words: Culture media. Helicobacter pylori. Metronidazole.
Introduction
Helicobacter pylori was first isolated from the gastric mucosa of patients with histological gastritis.' It has since been identified worldwide as a common infection involving the upper gastro-intestinal tract. It is the cause of type-B gastritis,2 plays a significant pathogenic role in duodenal and possibly gastric ulcers,3 and in gastric cancer.4 The organism is found on the gastric mucosa and in the gastric crypts throughout the stomach, and is protected from gastric acid, to which it is susceptible, by mucus. It may also occur in the duodenum, in association with metaplastic gastric mucosa.4
H. pylori is found throughout the world, but the incidence is much higher in developing countries where infection occurs early in childhood.' Over 80% of young adults may be infected in these areas, compared with a maximum of 40% in developed countries. Socio-economic status is a risk factor for infection.6 So far, the mode of transmission remains unclear.
H. pylori is a relatively difficult infection to treat, as the gastric habitat offers sanctuaries that partially shelter the organism from the topical or luminal effects of some antibiotics. H. pylori has also shown a tendency to acquire resistance rapidly to many classes of antibiotics given as a monotherapy. No current treatment will eradicate H. pylori in all cases. The two combinations most widely used are triple therapy or a combination of amoxycillin with omeprazole, and both have drawbacks. Triple therapy comprises colloidal bismuth subcitrate, tetracycline hydrochloride and metronidazole (Mz). This combination, taken for two weeks, provides eradication in 80-90% of cases. However, 3040% of patients experience side-effects which affect compliance.7
Resistance to Mz has considerable impact upon the success of triple therapy. In developing countries and among ethnic minorities where Mz is widely used to treat Giardia infections, resistance is endemic.8 This leads to a low cure rate when this therapy is used.
It is important for the laboratory to monitor current treatments. So far, only Mz resistance has been a problem and therefore it is important to test the susceptibility of the organism to this antibiotic, both before commencing treatment and in the case of treatment failure.
The minimum inhibitory concentration (MIC) agar dilution test is regarded as the best method for testing for antibiotic resistance,9 but it is uneconomical for small numbers of isolates. Most laboratories, therefore, use disc diffusion which correlates well with the MICs obtained by agar dilution.8 Recently, an Etest(R) (Cambridge Diagnostic Services, Cambridge, UK) has become available for Mz sensitivity,' and this is suitable for testing single isolates.
Our laboratory has reported a high level of Mz resistance: 80% compared to 30% in other epidemiological surveys.8 The aim of this study was to determine if this was due to laboratory practice, and, if so, to find an improved method for Mz sensitivity testing.
Materials and methods
Isolation of H. pylori and metronidazole susceptibility testing
At Birch Hill Hospital, Rochdale, endoscopy biopsies were sent to the microbiology laboratory in brain heart infusion broth (BHIB) (Lab M, Bury, UK) containing 0.4% yeast extract (Lab M) and 10% horse serum (Tissue Culture Services, Claydon, Bucks, UK). The specimens were inoculated onto Columbia blood agar (CBA) (Tissue Culture Services) containing haemin (X factor), and nicotinamide adenosine dinucleotide (V factor), 5% horse blood (Tissue Culture Services) and menadione. Two 5-gg Mz discs (Becton Dickinson, Oxford, UK) were added, one on the main inoculum and one on a thinner area of the inoculum. The plates were incubated in a microaerophilic atmosphere generated in an anaerobic jar11 using a gas pack (Oxoid Campylobacter System BRS6) and an anaerobic low-temperature catalyst (Oxoid BR42) (Oxoid, Basingstoke, Hants, UK). The jars were incubated at 35C for five days. The direct Mz sensitivity was interpreted from the initial culture, any zone being reported as sensitive.
Effect of culture media on metronidazole susceptibility
Two strains of H. pylori showing resistance to Mz on CBA with added X and V factors and menadione were sub-cultured onto fastidious anaerobe agar (FAA) (Lab M) containing 5% horse blood, and onto chocolate diagnostic sensitivity test agar (DSTA) (Lab M) containing 5% horse blood. It was found that both strains were susceptible to Mz on these media. Chocolate DSTA was difficult to read due to the translucentb nature of the colonies, whereas large zones of suceptibility were evident on FAA.
Effect of X factor, V factor and menadione on metronidazole susceptibility
Four strains of H. pylori were tested for Mz susceptibility on four different culture media using a standard inoculum (Macfarland's 4) (bioMerieux, Basingstoke, Hants, UK):
1 CBA without added factors
2 CBA with added V factor (12.5 (mu)g/L)
3 CBA with added X factor (12.5 (mu)g/L)
4 CBA with added menadione (2.5 (mu)g/L).
Four Mz discs were placed on the surface of the medium, and the cultures were incubated at 37C under micro-aerophilic conditions for 48 h. Bacteroides fragilis was used as a control. The zone sizes were measured.
Determination of optimum culture conditions
Nine strains of H. pylori were inoculated onto FAA, CBA and CBA with added X factor and menadione. These strains were incubated in four different ways:
1 An Mz disc (5 pg) was added, and the plates incubated micro-aerobically for 72 h.
2 An Mz disc (5 jig) was added and the plates preincubated anaerobically for 24 h, followed by 48 h in a micro-aerophilic atmosphere.
3 An Etest(R) strip was added and the plates incubated
micro-aerobically for 72 h.
4 An Etest(R) strip was added and the plates preincubated anaerobically for 24 h, followed by 48 h in a micro-aerophilic atmosphere.
Zone diameters around the discs were measured and MIC results were recorded from the Etest(R).
Comparison of metronidazole suscepitibility using disc diffusion and Etest(R) on fastidious anaerobe agar
Mz susceptibility of 48 strains of H. pylori was determined on FAA using Mz discs (5 jig) and Etest(R), using different incubation conditions:
1 Micro-aerophilic incubation for 72 h
2 Anaerobic pre-incubation for 24 h, followed by micro-aerophilic incubation for 48 h.
Statistical tests
Correlation between zone diameters and MICs determined by Etest(R) was calclated using the Instat statistical package.
Results
Effect of X factor, V factor and meNd:one on metronidazole susceptibility
The addition of X factor and menadione to CBA inhibited Mz susceptibility in two of the strains (2 and 3) tested (Table 1). This effect was peculiar to H. pylori. Bacteroides fragilis was fully sensitive to Mz on CBA supplemented with X factor, V factor and menadione (zone diameter: 35 mm).
Determination of optimum cultural conditions
When incubated for 72 h under micro-aerophilic conditions using 5 (mu)g Mz discs, eight out of nine strains (89%) showed total resistance to Mz on CBA with X factor and menadione, compared to three out of nine strains (33%) when incubated under the same conditions on FAA and CBA without added factors (Table 2). In all strains, the MIC determined by Etest(R) was >32 mg/L when grown on CBA with added X factor and menadione. Only three out of nine strains (33%) showed the same MIC on CBA and FAA.
Twenty-four hour anaerobic pre-incubation of the plates resulted in increased zone sizes in six out of nine strains (66%) on FAA and CBA, and two out of nine strains (22%) when grown on CBA with added X factor and menadione. Twenty-four hour anaerobic pre-incubation resulted in a decrease in the MIC of Mz in four out of nine strains (44%) and an increase in the MIC in two out of nine strains (22%) on FAA. There was a decrease in MIC in six out of nine strains (66%) on CBA and a decrease in MIC in three out of nine strains (33%) when grown on CBA with added X factor and menadione.
Comparison of metronidazole susceptibility using disc diffusion and Etest(R) on fastidious anaerobe agar
The relationship between zone diameter and MIC obtained using the Etest(R) was correlated. The results obtained after 72 h micro-aerophilic incubation gave a correlation coefficient (r) of 0.9518 (Instat statistical package). When the same strains were pre-incubated under anaerobic conditions for 24 h, followed by 48 h micro-aerophilic incubation, the correlation coefficient (r) was 0.7119. In addition, anaerobic preincubation for a 24 h period resulted in increased zone diameter and MIC.
Of the 48 strains tested on FAA, 29 (60.4%) were sensitive to Mz and 19 (39.6%) were resistant. Previous results of 58 strains identified in the laboratory, using CBA with added X factor, V factor and menadione, showed that 11 (19%) were sensitive to Mz and 37 (64%) were resistant.
Discussion
H. pylon is usually treated by combination therapy, but an increasing level of Mz resistance2 has led to routine laboratory testing of Mz susceptibility.
At Birch Hill Hospital, Rochdale, the rate of Mz resistance prior to this study was 80%, which was higher than incidence rates reported by other hospitals. Other workers have used CBA for Mz sensitivity testing of H. pylori8,13 and have reported lower rates of Mz resistance. Two strains of H. pylori which had previously appeared to be Mz-resistant, were selected and found to be Mz-sensitive on DSTA and FAA. Although H. pylori grew on the chocolate DSTA, the zone edge was difficult to determine. The zone edge was easier to distinguish on FAA medium.
The CBA used routinely in our laboratory included both X and V factors plus menadione, added to the basic medium as additional growth factors. As other workers using CBA have not reported a high level of Mz resistance, the effect of these additional factors on Mz susceptibility was investigated. It was found that the six strains which would have been reported as sensitive to Mz using FAA without pre-incubation were all found to be resistant when X factor and menadione were added to the medium. It is important to note that four of the sensitive strains showed smaller zone diameters around the Mz disc on FAA than on CBA without X and V factors. FAA contains small quantities of added X factor, and this could account for the slight inhibition seen.
Once it was established that the CBA used was unsuitable for testing Mz sensitivity of H. pylori, a new laboratory protocol was introduced. This included anaerobic pre-incubation and micro-aerophilic incubation to compare the results obtained using a greater number of strains. Mz disc and Etest(R) were also tested in parallel. There was good correlation between zone diameter and MIC, and there was an increase in the level of sensitivity when anaerobic pre-incubation was included. It was decided to use disc sensitivity testing in preference to Etest(R) to reduce cost. Anaerobic pre-incubation of the plates gave larger zone sizes, but there remains some doubt about its accuracy, and its routine use is not recommended until further work is carried out.
Anaerobic pre-incubation for 24 h increased zone diameter and MIC in some of the nine strains, and this depended upon the medium used. In some cases, the difference was sufficient to change the result from resistant to sensitive, and in almost every case there was a significant increase in susceptibility. Although anaerobic pre-incubation affects Mz susceptibility in vitro, further work is needed to relate these effects to the clinical outcome.
Overall, using FAA as a sensitivity medium under micro-aerophilic conditions, the percentage of resistant strains was reduced to 39.6% which compared well with the rest of the western world (15-35% resistants). This figure may have been reduced further if a sensitivity medium without any added X factor was used.
As a result of this work, the following protocol for the culture of biopsy specimens and the Mz sensitivity testing of H. pylori is suggested: Culture all biopsies on CBA and a selective medium containing antibiotics. Incubate under micro-aerophilic conditions for 72 h. Test suspect colonies for urease. Inoculate all positive isolates into tryptone water to a concentration equivalent to McFarland's standard Number 3, and lawn CBA without additional X factor, V factor and menadione. Place a 5 (mu)g Mz disc in the centre and incubate microaerophilically for 72 h. Perform catalase and oxidase tests and a Gram stain of the colony to identify the organism as H. pylori. Sensitivity is indicated by a zone of 15 mm or more around the Mz disc. It is essential that additional factors are not added to media used for sensitivity testing.
[Reference]
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[Author Affiliation]
KATHLEEN J. TAYLOR, VALERIE EDWARDS-JONES* AND MAVIS ARMITAGE
Department of Microbiology, Birch Hill Hospital, Rochdale, Lancashire OL12 9QB and *Department of Biological Sciences, Manchester Metropolitan University, Chester Street, Manchester M1 5GD, England, UK
Accepted 17 October 1997)
[Author Affiliation]
Correspondence to Kathleen Taylor
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