Advances in Microbiology, 2012, 2, 263-267
http://dx.doi.org/10.4236/aim.2012.23031 Published Online September 2012 (http://www.SciRP.org/journal/aim)
Determination of Antibiotic Sensitivity Pattern of
Helicobacter pylori Isolates from South India Population
by Epsilometer Test (E-Test)
Khawaja Shakeel Ahmed1*, Anghesom Ambesajir Ghebremedhin1, Aleem Ahmed Khan2,
Santosh K. Tiwari2, J. D. Ahi3, Irshad Ahmed2
1Microbiology Unit, Department of Clinical Laboratory Sciences, Asmara College of Health Sciences, Asmara, Eritrea
2Centre for Liver Research and Diagnostics, Hyderabad, India
3Dr. Hari Singh Gour University, Sagar, India
Received March 15, 2012; revised April 5, 2012; accepted April 24, 2012
Introduction: The importance of H. pylori as an etiological agent in gastroduodenal disease had suggested antibiotic
treatment as a main target for the elimination of infection. The successful eradication of H. pylori infection was shown
to resolve the gastritis, dramatically accelerate ulcer healing, reduce ulcer recurrence and the prophylactic effect on the
recurrence of ulcer bleeding. Materials and Methods: In the present study a total six antimicrobial agents such as
Ciprofloxacin, Metronidazole, Norfloxacin, Tetracyclin, Amoxycillin and Clarithromycin were used against 100 clinical
isolates from patients with peptic ulcer, and non-ulcer dyspepsia. H. pylori was isolated from the gastric biopsies ob-
tained from the clinical subjects. Results: Results of our study showed 100% resistance to Metronidazole (MIC > 256
μg/ml). 97% of the isolates were observed sensitive to Ciprofloxacin (MIC 0.38 μg), whereas Tetracyclin showed 96%
sensitivity (MIC 0.25 μg - 0.125 μg). Resistance to amoxycillin, Norfloxacin, and Clarithromycin were 80%, 38% and
76% respectively (MIC > 256 μg/ml). Conclusion: It is observed that 96% - 97% of the clinical isolates were sensitive
to Tetracycline and ciprofloxacin, where all isolates were found to be resistant to Metronidazole. Resistance to Amoxy-
cillin and Clarithromycin were 80% and 76% respectively. Studies attributed the high level of resistance to the frequent
use of the drugs to treat various other infections and ineffective drug control policy.
Keywords: Helicobacter pylori; Culture; Metronidazole; Tetracycline; Clarithromycin
The importance of H. pylori as an etiological agent in
gastroduodenal disease had suggested antibiotic treat-
ment as a main target for the elimination of infection.
The successful eradication of H. pylori infection was
shown to resolve the gastritis, dramatically accelerate
ulcer healing and reduce ulcer recurrence and the pro-
phylactic effect of the recurrence of ulcer bleeding .
Several studies were carried out to find an effective and
well tolerated antibiotic regime for the treatment of H.
pylori associated diseases. Despite the common avail-
ability of various treatment regimes for H. pylori infec-
tion, eradication regime of choice has not yet been ac-
cepted. Few studies suggested that combined antibiotic
regime with two antibiotics and one acid suppressive
agent to be effective but its efficacy was observed to vary
significantly when the regime was tested in different
geographical regions, suggesting a need for a universally
effective antibiotic regime of choice. The most com-
monly used antibiotics in combination therapies world-
wide are Metronidazole, Ciprofloxacin, Amoxycillin,
Clarithromycin, and Tetracyclin. But the antibiotic sensi-
tivity pattern against these antimicrobials from various
geographical regions shows diversified results particu-
larly from the developing countries. Studies ascribed
several factors that could lead to the treatment failure .
Presence of resistant organism or acquiring resistance to
the antibiotics are the most important variables for an
observed diversified cure rate [3,4]. Bacterial heteroge-
neity , dietary habits could also be shown to contribute
significantly in several Asian and European countries,
where resistance to antibiotics particularly for the Met-
ronidazole and Clarithromycin through out 1990s. It is
reported to vary between 10% - 80% among the different
geographic regions [6,7]. Mollison et al. 1998  re-
ported 36% and 11% resistance to Metronidazole and
Clarithromycin. Alarcon et al. (1999)  determined the
opyright © 2012 SciRes. AiM
K. S. AHMED ET AL.
frequency of resistance to Metronidazole, tetracycline,
Amoxyicilin and Clarithromycin in 282 H. pylori clinical
isolates from spain and evaluated the evolution of resis-
tance over five years of their study. Data infers a steady
increase in the metronidazole resistance from 9% to 33%.
Similarl increase of resistance to Clarithromycin and
Tetracycline was observed. Mendonca et al. (2000) 
reported 42% resistance to Metronidazole, 29% to Amo-
xycillin and 7% for Clarithromycin and tetracycline.
Similarly Mijaji et al. 1997  reported a very high
acquired resistance to Metronidazole 66.7% and 70.6%
for Clarithromycin after the dual and triple therapy. In
India studies reported a relatively high resistance to
Metronidazole and Clarithromycin i.e. between 90% -
100% and 30 - 50 respectively . Resistance to Amo-
xycillin was reported to be 40%. Thus as a result of
changing patterns of antibiotic sensitivity between dif-
ferent geographic regions and also within the same re-
gion it is suggested valuable to test the sensitivity regu-
larly before deciding the therapeutic regime.
Additional limitations to the treatment failure could be
the inaccessibility of the antibacterial agent to the organ-
ism residing deep at the inter cellular junction, or inside
the gastric mucous cell hence it re-establishes the infec-
tion after the treatment, as suggested by the transmission
electron microscopy and similar DNA patterns of the H.
pylori strains before and after treatment [12,13]. The
bioavailibity and stability of the drug at gastric pH is an
important factor for determining the choice of eradication
therapy. Metronidazole is very stable at pH ranging from
2.0 - 7.0 but increasing prevalence of pretreatment resis-
tant strain hampered its use as a drug of choice . Re-
sistance to Amoxycillin though not reported commonly,
the drug is less stable particularly at low pH. Clarithro-
mycin is the most acid labile drug commonly used to
eradicate the H. pylori. However significant increase in
the efficacy of these drugs is observed by the co-admini-
stration of Proton Pump Inhibitors . Thus in vitro
antimicrobial susceptibility test helps in predicting the
clinical response to treatment and prevalence of antibi-
otic resistant strains between the different geographic
regions and also among the sub groups within the study
population and guide the selection of antibiotics.
The Minimum Inhibitory Concentration (MIC) of an
organism is the lowest concentration of an antibiotic that
will inhibit its growth. Bacteria are classified as sensitive,
intermediate or resistant based on the breakpoint MIC
values that are arbitrarily defined and reflect the achiev-
able levels of the antibiotic, the distribution of MIC’s for
the organism and their correlation with clinical outcome.
Disk diffusion, agar dilution and Epsilometer test (E-
Test), where the two fold serial dilution of antibiotics
were in-corporated in to tubes of broth, agar plates or on
a paper strip respectively, are different methods to meas-
ure the MIC of the organism.
The Epsilometer test (E-Test AB Biodisk, solna, Swe-
den) is a recently developed technique for the quantita-
tive determination of susceptibility to antimicrobial agents
in a variety of bacteria and fungi . The test is based
on the combination of concepts of both the agar dilution
and disk diffusion methods but differs from the conven-
tional disk diffusion method by the use of the predefined
exponential gradient of antibiotics with the concentration
minimum (least concentration) and the concentration
maximum (highest concentration). The gradients cover a
continuous concentration range depending on the antibi-
otic, which corresponds to 15 two old dilution in a con-
ventional MIC method. The E test is also much less labor
intensive and is easier to perform than agar dilution and
broth dilution method, which also allows the test to be
quickly and economically adapted into the laboratory
The MIC value is read from the scale in terms of
μg/ml where the ellipse edge intersects the strips. Studies
have assayed several antimicrobials commonly used to
treat infection using E-Test and reported a good correla-
tion with standard methods. The resistance breakpoint
used for is Metronidazole > 8 μg, Clarithromycin > 1 μg,
Tetracyclin > 2 μg, Amoxycillin > 8 μg, Norfloxacin >
8.0 μg and Ciprofloxacin > 1.0 μg .
The Present study was designed to determine the sen-
sitivity pattern of various commonly used antimicrobial
agents to treat the infection. In this study a total of six
antimicrobial agents such as Ciprofloxacin, Metronida-
zole, Norfloxacin, Tetracyclin, Amoxycillin and Clari-
thromycin were tested by E-Test against 100 clinical
isolates from patients with peptic ulcer, and non-ulcer
dyspepsia. Having established the prevalence of H. pylori
infection, patients are currently treated (with a view to
eradication) with combination therapy (amoxicillin, cipro-
floxacin and a proton pump inhibitor/bismuth). The rela-
tionship between antimicrobial resistance and the suc-
cessful treatment of H. pylori infection has dictated that a
study of the incidence of acquired antimicrobial resistance
among H. pylori isolates obtained within our environ-
ment be determined hence the importance of this study.
2. Materials and Methods
2.1. Media Preparation
1.4 gm of Brucella Agar media was dissolved in 100 ml
of distilled water and the media was autoclaved at 120
lbs for 15 minutes. The medium was supplemented with
7% sheep blood.
In the present study a total of six antimicrobial agents
Copyright © 2012 SciRes. AiM
K. S. AHMED ET AL. 265
such as Ciprofloxacin, Metronidazole, Norfloxacin, Tet-
racyclin, Amoxycillin and Clarithromycin were used
against 100 clinical isolates from patients with peptic
ulcer, and non-ulcer dyspepsia. H. pylori was isolated
from the gastric biopsies obtained from the clinical sub-
jects. After the primary identification of the bacterium by
the conventional biochemical method, bacteria were
subcultured on the brucella agar plates supplemented
with sheep blood. Colonies were suspended in 1 ml of
Brucella broth to achieve turbidity equivalent to that of
no 3 McFarland turbidity standard. 140 mm diameter
agar plates were inoculated by swabbing of the surface.
The plates were incubated at 37˚C under microaerophillic
conditions. MICs were read after 48 hrs of incubation on
the basis of the intersection of the elliptical zone of the
growth inhibition with the MIC scale on the E-Test strip
The 100 H. pylori culture isolated from Peptic Ulcer and
Non-Ulcer Dyspepsia patients were tested for their an-
timicrobial sensitivity by E-Test with six antimicrobials.
Results of our study showed 100% resistance to Met-
ronidazole (MIC > 256 μg/ml). 97% of the isolates were
observed to be sensitive to Ciprofloxacin (MIC 0.38 μg),
whereas Tetracycline showed 96% sensitivity (MIC 0.25
μg - 0.125 μg). Resistance to amoxycillin, Norfloxacin,
and Clarithromycin were 80%, 38% and 76% respec-
tively (MIC > 256 μg/ml) (Table 1).
In vitro sensitivity testing of H. pylori is regarded as an
important test as no regimen is proved to be universally
successful. Sensitivity profile is recommended to be de-
termined regionally and periodically before starting the
treatment. In this study six commonly used antibiotics
were tested by E-Test against 100 clinical H. pylori iso-
lates obtained from different diseased group. Our data on
100 clinical isolates showed 95% - 97% sensitivity to
Ciprofloxacin (MIC-0.38 μg) and Tetracycline (MIC-
Figure 1. Photograph showing the sensitivity and resistance
towards different antibiotics by Helicobacter pylori.
Table 1. Sensitivity of Helicobacter pylori isolates (n = 100)
to different antimicrobial agents.
Agent Tested% of Isolates
Inhibited % of Isolates
Amoxyicilin 16% = 0.25 mcg 4% = 32.0 mcg 80%
Ciprofloxacin97% = 0.38 mcg - 3%
Tetracycline 66% = 0.25 mcg 30% = 0.125 mcg4%
Norfloxacin 40% = 1.5 mc g 12% = 4.0 mcg 38%
Clarithromycin10% = 8.0mcg 16% = 48.0 mcg 76%
Metronidazole- - 100%
0.25 μg and 0.125 μg) and all the clinical isolates were
found to be resistant to Metronidazole (MIC < 256 μg).
Resistance to Clarithromycin, Amoxycillin, and Nor-
floxacin is 76%, 80% and 38% respectively (MIC < 256
High resistance observed for Metronidazole could be
due to the frequent use of the antibiotic in the empiric
treatment of diarrhea . The use of Metronidazole for
dental infections may also add to selection pressure. In
addition antibiotics self-medication is encouraged by free
access and over the counter purchase and by ineffective
drug control policy. This could be a contributing factor
for the very high level of resistance of H. pylori to
Amoxyicilin (80%), Clarithromycin (76%), Metronida-
zole (100%) . Though these data sets are difficult to
directly compare, it appears that Metronidazole and amo-
xicillin resistance have remained relatively stable while
Clarithromycin resistance has increased. The increasing
background rate of Clarithromycin resistance provides at
least a partial explanation for the decreasing efficacy of
traditional Clarithromycin-containing regimens. It is
quite clear that Clarithromycin resistance, which has been
attributed to several different point mutations in the pep-
tidyltransferase region encoded in domain V of the 23S
rRNA gene (142), is associated with a high rate of treat-
ment failure when Clarithromycin containing regimens
are employed (148 - 150) .
In one study, scientist determined the frequency of re-
sistance to Amoxycillin, Tetracycliin, Metronidazole and
Clarithromycin in 282 H. pylori isolates from Spain and
evaluated the evolution of resistance over five years of
the study. The overall percentage of resistance for Met-
ronidazole was 19.9% and 3.5% for Clarithromycin.
They observed a steady increase in resistance to met-
ronidazole from 9% in 1991 to 21.6% in 1995. No
Amoxycillin and Tetracyclin resistance was observed in
the strains tested against Metronidazole, Tinidazole and
Clarithromycin. Resistance to Amoxycillin is 73%. How-
ever a comparatively lower resistance was observed at
lucknow, 66% for Metronidazole and 28% for Ampicillin
Copyright © 2012 SciRes. AiM
K. S. AHMED ET AL.
. This difference in the sensitivity pattern observed in
western as well as in Asian isolates could be due to the
frequent use of the antibiotics to treat other infections.
Moreover, ineffective drug control policy also has a con-
tribution. Another Possibility could be due to infection
with different strains of H. pylori.
Results of our study support that Tetracyclin and
Ciprofloxacin could be the antibiotics of choice in the
eradication of H. pylori. It would be necessary to inves-
tigate ciprofloxacin in any eradication treatment regime
in our setting, since it appears to be the most active anti-
biotic in eradicating H. pylori in this environment.
It is observed that 96% - 97% of the clinical isolates were
sensitive to Tetracycline and Ciprofloxacin, where all
isolates were found to be resistant to Metronidazole. The
same resistance to metronidazole has also been reported
from other geographic regions. Resistance to Amoxycil-
lin and Clarithromycin were 80% and 76% respectively.
Studies attribute the high level of resistance to the fre-
quent use of the drugs to treat various other infections,
ineffective drug control policy and infection with differ-
ent strains of H. pylori. The relationship between antim-
icrobial resistance and the successful treatment of H.
pylori infection has dictated that a study of the incidence
of acquired antimicrobial resistance among H. pylori
isolates obtained within our environment be determined
hence the importance of this study. The ministry of
health should have an effective drug control policy. Fur-
thermore, there is the need to continue the evaluation of
new treatment agents such as NE-2001 , older agents
such as nitrofurantoin  or introduction of herbal
management in order to eradicate Helicobacter pylori.
Future case-control studies employing larger sample size
are needed to demonstrate the effectiveness or otherwise
of therapy in the different patient groups. A long-term
follow-up of our patients will also contribute to the de-
velopment of guidelines on the issues of referral, diag-
nostic methods and treatment of H. pylori.
We would like to thank our colleagues at our department
for their fruitful discussion during the research and their
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