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Antimicrobial Agents and Chemotherapy, June 1999, p. 1484-1486, Vol. 43, No. 6
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Subpopulations of Helicobacter pylori
Are Responsible for Discrepancies in the Outcome of Nitroimidazole
Susceptibility Testing
E. J.
van der
Wouden,1
A.
de
Jong,2
J. C.
Thijs,1
J. H.
Kleibeuker,3 and
A. A.
van Zwet2,*
Department of Internal Medicine, Bethesda
Hospital,1 and Regional Public Health
Laboratory Groningen/Drenthe,2 Hoogeveen, and
Department of Gastroenterology, University Hospital,
Groningen,3 The Netherlands
Received 12 October 1998/Returned for modification 15 January
1999/Accepted 20 March 1999
 |
ABSTRACT |
Metronidazole susceptibility testing by E test was compared to that
by disk diffusion for 263 Helicobacter pylori isolates and
to that by breakpoint agar dilution for 90 H. pylori
isolates. In 5% and 6% of the cases, respectively, results were
discrepant. For each of 52 clinical isolates an E test was performed on
10 separate colonies. Subpopulations of resistant and susceptible bacteria were found in five cases. From three isolates, each colony was
subcultured and tested up to 10 times. All but 1 of 292 tests showed
the same result. We conclude that the E test is reliable and that
subpopulations are responsible for discordant results.
| |
TEXT |
Nitroimidazoles are used to treat
Helicobacter pylori infection (22). The relevance
of in vitro nitroimidazole resistance (NIR) of H. pylori to
treatment efficacy is still debated (2, 5, 17, 20, 24).
Furthermore, data on the evolution of NIR prevalence are conflicting
(6, 14-16, 23). As nitroimidazole susceptibility testing is
not standardized, methodology may be at least partly responsible for
these controversies (17). In addition, susceptibility
testing may also be influenced by the fact that patients can be
infected with both susceptible and resistant bacteria (25).
In this study the relevance of this phenomenon to E-test results was evaluated.
Antral biopsy specimens were rubbed on two types of selective agar
plates, one containing Belo-horizonte medium (BHM medium) and the other
containing campylobacter-selective medium. The plates were incubated at
36°C under microaerophilic conditions and were examined after 2 to 3 days. Colonies were confirmed to be H. pylori by Gram
staining and determination of enzyme activities (catalase, oxidase, and
urease). For susceptibility testing, Columbia agar plates, supplemented
with 7% horse blood but without antibiotics (H. pylori
susceptibility testing medium [HPS medium]), were inoculated with a
suspension of multiple colonies. Metronidazole susceptibility was
tested by E test, disk diffusion method, and breakpoint agar dilution
method. The plates were read after 2 to 3 days. For the E test (AB
Biodisk, Solna, Sweden) strains were considered resistant if the MIC of
metronidazole was above 8 µg per ml. For disk diffusion a 5-µg
metronidazole disk (Mast Laboratories, Liverpool, United Kingdom) was
used. Strains with an inhibition zone of less than 10 mm were regarded
as resistant (19). For breakpoint agar dilution two types of
plates, one containing HPS medium supplemented with metronidazole (8 µg per ml) and the other containing HPS medium without antibiotics as
a control, were used. If H. pylori grew on both plates, the
strain was considered resistant.
In the first part of the study, the E-test results were compared with
those of disk diffusion for 263 consecutive H. pylori isolates and with those of breakpoint agar dilution in 90 other isolates. All isolates were obtained from different patients. In the
second part, 52 isolates, each obtained from a single antral biopsy
specimen from 1 of 52 patients, were investigated. From each isolate 10 to 11 separate colonies were subcultured, and an E test was performed
on each subculture. Results were compared with the E-test results
obtained by using multiple colonies. For 3 of these 52 isolates (2 with
MIC of 
256 µg per ml and 1 with MIC of 0.5 µg per ml as
determined by E test with multiple colonies), the 10 to 11 colonies
were also subcultured on BHM medium. Bacteria were harvested after 2 to
3 days and resuspended on HPS medium for the E test or on BHM medium
for further subculture. After 2 to 3 days, bacteria were again
harvested from the BHM medium and the same procedure was repeated. In
this way, 32 colonies were each tested up to 10 times.
When the E-test results were compared to the disk diffusion results and
breakpoint agar dilution results, discordance was found in 5% (95%
confidence interval [CI], 3 to 9%) and 6% (95% CI, 2 to 12%) of
cases, respectively: for the comparison between E test and disk
diffusion, 166 strains were susceptible by both tests, 83 strains were
resistant by both tests, 8 strains were resistant by E test but
susceptible by disk diffusion, and 6 strains were susceptible by E test
but resistant by disk diffusion; for the comparison between E test and
breakpoint agar dilution, 60 strains were susceptible by both tests, 25 strains were resistant by both tests, 1 strain was resistant by E test
but susceptible by agar dilution, and 4 strains were susceptible by E
test but resistant by agar dilution. In most of the cases of
discordance the differences between the E-test and disk diffusion
results were not around the cutoff: bacteria were completely resistant by one test and fully susceptible by the other test (Table
1). When 10 to 11 different colonies of
an isolate were studied, 34 biopsy specimens were found to harbor only
susceptible bacteria and in 13 specimens only resistant bacteria were
found. In all of these cases the results were congruent with the
outcome of routine susceptibility testing performed by using multiple
colonies. In five biopsy specimens (10%; 95% CI, 3 to 21%) both
resistant and susceptible bacteria were found. When multiple colonies
were used, three of these five isolates were found to be resistant and
the other two were found to be susceptible (Table
2). In our last study, in which the E
test was repeated up to 10 times for each of 32 single colonies
obtained from three isolates, variations in MIC were frequently seen.
For one strain, for example, the MIC varied between 0.032 and 2 µg
per ml. However, only one strain that was initially designated as
resistant (for which the MIC varied between 24 and 256 µg per ml)
was reclassified as susceptible (MIC was 8 µg per ml) (1 of 292 tests, 0.34%; 95% CI, 0.01 to 1.84%).
View this table:
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[in a new window]
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TABLE 1.
MIC and inhibition zones of the 14 strains with
discordant results in the study comparing E test to disk diffusion
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Results of the E test performed on 10 to 11 separate
colonies as compared to the E test performed on multiple colonies for
the five biopsy specimens containing both resistant bacteria and
susceptible bacteria
|
|
It has generally come to be recognized that, in comparison with testing
of H. pylori susceptibility to other antibiotics, testing of
nitroimidazole susceptibility is problematic (3, 9, 10, 17,
21). In our study we found disagreement between the tests in
approximately 5% of cases, as did Midolo et al. (18). Although differences in methodology may cause variations in test results (17), we think that this does not account for the
discrepancies found in our study, namely, that isolates were classified
as highly resistant by one test and fully susceptible by another test.
As the reproducibility of the E test for testing of the offspring of a
single bacterium was almost perfect, the most likely explanation for
the discordant results is a coinfection with both resistant bacteria
and susceptible bacteria.
In our population such a coinfection could be demonstrated in
approximately 10% of the cases, when only one biopsy specimen was
examined. This is not unexpected, as it is known from DNA fingerprinting that patients can be infected with two or more different
strains (8, 11, 13). Moreover, even within the same strain
different patterns of susceptibility to metronidazole have been found
(7, 13). As a study in gnotobiotic piglets has shown a
microclonal mode of growth with limited migration of bacteria between
different sites of the stomach (1), it is likely that as the
number of gastric biopsy specimens examined increases the greater will
be the number of different subpopulations encountered (13).
It is remarkable that coinfections with both resistant bacteria and
susceptible bacteria have not been described for other antibiotics
(7). The special position of nitroimidazoles in this respect
may be explained by the genetic basis of NIR (9). NIR is
related to null mutations in the rdxA gene, which encodes a
nitroreductase. This enzyme is not essential for bacterial survival, as
no decrease in metabolic or growth capacity is observed in the absence
of functional enzyme (12). When a nitroimidazole-containing therapy fails to eradicate H. pylori, resistant mutants will
become the major population in the stomach. After the antibiotic
pressure has been removed, the wild type may reappear, but it may have no significant survival advantage. This results in a stable coinfection with both resistant bacteria and susceptible bacteria. In contrast, clarithromycin resistance is associated with a point mutation in rRNA
genes (4). Inefficient protein synthesis, due to the mutation, is likely to diminish the chances of survival in the absence
of clarithromycin. Therefore, if clarithromycin-susceptible bacteria
are present they will eventually constitute the majority of the population.
In conclusion, our study shows that the E test is reliable for
nitroimidazole susceptibility testing of H. pylori. A
coinfection with both resistant bacteria and susceptible bacteria,
however, is not uncommon and may lead to discordance between the
results of different tests.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Regional Public
Health Laboratory Groningen/Drenthe, Bethesda Hospital, Dr. G. H. Amshoffweg 1, 7909 AA Hoogeveen, The Netherlands. Phone: 31 (0)
528-286222. Fax: 31 (0) 528-286435. E-mail: aavzwet{at}wxs.nl.
| |
REFERENCES |
| 1.
|
Akopyants, N. S.,
K. A. Eaton, and D. E. Berg.
1995.
Adaptative mutation and cocolonization during Helicobacter pylori infection of gnotobiotic piglets.
Infect. Immun.
63:116-121[Abstract].
|
| 2.
|
Bateson, M. C.
1997.
Helicobacter pylori eradication.
Aliment. Pharmacol. Ther.
11:1001[Medline]. (Letter.)
|
| 3.
|
Cederbrant, G.,
G. Kahlmeter, and A. Ljungh.
1993.
The E test for antimicrobial susceptibility testing of Helicobacter pylori.
J. Antimicrob. Chemother.
31:65-71[Abstract/Free Full Text].
|
| 4.
|
Debets-Ossenkopp, Y. J.,
M. Sparrius,
J. G. Kuster,
J. J. Kolkman, and C. M. J. E. Vandenbroucke-Grauls.
1996.
Mechanisms of clarithromycin resistance in clinical isolates of Helicobacter pylori.
FEMS Microbiol. Lett.
142:37-42[Medline].
|
| 5.
|
De Boer, W. A., and G. N. J. Tytgat.
1996.
How to treat Helicobacter pylori infection should treatment strategies be based on testing bacterial susceptibility? A personal viewpoint.
Eur. J. Gastroenterol. Hepatol.
8:709-716[Medline].
|
| 6.
|
De Koster, E.,
A. Cozzoli,
C. VandenBorre,
C. Jonas,
P. Denis,
J. Otero, and M. Deltenre.
1997.
HP resistance to macrolides increases, to imidazoles remains stable.
Gastroenterology
112:A99. (Abstract.)
|
| 7.
|
Dore, M. P.,
M. S. Osato,
D. H. Kwon,
D. Y. Graham, and F. A. K. El-Zaatari.
1998.
Demonstration of unexpected antibiotic resistance of genotypically identical Helicobacter pylori isolates.
Clin. Infect. Dis.
27:84-89[Medline].
|
| 8.
|
Fujimoto, S.,
B. Marshall, and M. J. Blaser.
1994.
PCR-based restriction fragment length polymorphism typing of Helicobacter pylori.
J. Clin. Microbiol.
32:331-334[Abstract/Free Full Text].
|
| 9.
|
Goodwin, A.,
D. Kersulyte,
G. Sisson,
S. J. O. Veldhuyzen van Zanten,
D. E. Berg, and P. S. Hoffman.
1998.
Metronidazole resistance in Helicobacter pylori is due to null mutations in a gene (rdxA) that encodes an oxygen-insensitive NADPH nitroreductase.
Mol. Microbiol.
28:383-393[Medline].
|
| 10.
|
Hachem, C. Y.,
J. E. Clarridge,
R. Reddy,
R. Flamm,
D. G. Evans, and D. Y. Graham.
1995.
There are discrepancies between the results of antimicrobial susceptibility testing of Helicobacter pylori using the E-test compared to broth microdilution or disk diffusion.
Gastroenterology
108:A106. (Abstract.)
|
| 11.
|
Hirschl, A. M.,
M. Richter,
A. Makristathis,
P. M. Pruckl,
B. Willinger,
K. Schutze, and M. L. Rotter.
1994.
Single and multiple strain colonization in patients with Helicobacter pylori associated gastritis: detection by macrorestriction DNA analysis.
J. Infect. Dis.
170:473-475[Medline].
|
| 12.
|
Hoffman, P. S.,
A. Goodwin,
J. Johnsen,
K. Magee, and S. J. O. Veldhuyzen van Zanten.
1996.
Metabolic activities of metronidazole-sensitive and -resistant strains of Helicobacter pylori: repression of pyruvate oxidoreductase and expression of isocitrate lyase activity correlate with resistance.
J. Bacteriol.
178:4822-4829[Abstract/Free Full Text].
|
| 13.
|
Jorgensen, M.,
G. Daskalopoulos,
V. Warburton,
H. M. Mitchell, and S. L. Hazell.
1996.
Multiple strain colonization and metronidazole resistance in Helicobacter pylori-infected patients: identification from sequential and multiple biopsy specimens.
J. Infect. Dis.
174:631-635[Medline].
|
| 14.
|
Karim, Q. N., and R. P. H. Logan.
1996.
Helicobacter pylori (H. pylori) antimicrobial resistance in the UK.
Gut
39:A15[Free Full Text].
|
| 15.
|
Ling, T. W. K.,
A. F. B. Cheng,
J. J. Y. Sung,
P. Y. L. Yiu, and S. S. C. Chung.
1996.
An increase in Helicobacter pylori strains resistant to metronidazole: a five-year study.
Helicobacter
1:57-61[Medline].
|
| 16.
|
Lopez-Brea, M.,
D. Domingo,
I. Sanchez, and T. Alcaron.
1998.
Evolution of resistance to metronidazole and clarithromycin in Helicobacter pylori clinical isolates from Spain.
J. Antimicrob. Chemother.
40:279-281[Abstract/Free Full Text].
|
| 17.
|
Megraud, F.
1997.
Resistance of Helicobacter pylori to antibiotics.
Aliment. Pharmacol. Ther.
11(Suppl. 1):43-53.
|
| 18.
|
Midolo, P. D.,
J. M. Bell,
J. R. Lambert,
J. D. Turnidge, and M. L. Grayson.
1997.
Antimicrobial resistance testing of Helicobacter pylori: a comparison of E-test and disk diffusion methods.
Pathology
29:411-414[Medline].
|
| 19.
|
Midolo, P. D.,
J. Turnidge,
J. R. Lambert, and J. M. Bell.
1995.
Validation of a modified Kirby-Bauer disk diffusion method for metronidazole susceptibility testing of Helicobacter pylori.
Diagn. Microbiol. Infect. Dis.
21:135-140[Medline].
|
| 20.
|
Misiewicz, J. J.
1997.
Management of Helicobacter pylori-related disorders.
Eur. J. Gastroenterol. Hepatol.
9(Suppl. 1):17-21.
|
| 21.
|
Piccolomini, R.,
G. Di Bonaventura,
G. Catamo,
F. Carbone, and M. Neri.
1997.
Comparative evaluation of the E test, agar dilution, and broth microdilution for testing susceptibilities of Helicobacter pylori strains to 20 antimicrobial agents.
J. Clin. Microbiol.
35:1842-1846[Abstract].
|
| 22.
|
Van der Hulst, R. W. M.,
J. J. Keller,
E. A. J. Rauws, and G. N. J. Tytgat.
1996.
Treatment of Helicobacter pylori infection: a review of the world literature.
Helicobacter
1:6-19[Medline].
|
| 23.
|
Van der Wouden, E. J.,
A. A. van Zwet,
J. C. Thijs,
G. D. C. Vosmaer,
J. A. J. Oom,
A. de Jong, and J. H. Kleibeuker.
1997.
Rapid increase in the prevalence of metronidazole resistant Helicobacter pylori.
Emerg. Infect. Dis.
3:385-389[Medline].
|
| 24.
|
Walt, R. P.
1996.
Metronidazole resistant H. pyloriof questionable clinical importance.
Lancet
348:489-490[Medline].
|
| 25.
|
Xia, H. H. X.,
J. Kalantar, and N. J. Talley.
1998.
Metronidazole- and clarithromycin-resistant Helicobacter pylori in dyspeptic patients in western Sydney as determined by testing multiple isolates from different gastric sites.
J. Gastroenterol. Hepatol.
13:1044-1049[Medline].
|
Antimicrobial Agents and Chemotherapy, June 1999, p. 1484-1486, Vol. 43, No. 6
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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