Determination of Antibiotic Sensitivity Pattern of <i>Helicobacter pylori</i> Isolates from South India Population

doi:10.4236/aim.2012.23031

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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

Email: *[email protected]

Received March 15, 2012; revised April 5, 2012; accepted April 24, 2012

ABSTRACT

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

1. Introduction

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 [1].

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 [2].

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 [5], 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 [8] re-

ported 36% and 11% resistance to Metronidazole and

Clarithromycin. Alarcon et al. (1999) [6] determined the

*Corresponding author.

K. S. AHMED ET AL.

264

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) [9]

reported 42% resistance to Metronidazole, 29% to Amo-

xycillin and 7% for Clarithromycin and tetracycline.

Similarly Mijaji et al. 1997 [10] 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 [11]. 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 [14]. 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 [15]. 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 [16]. 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

work flow.

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 [17].

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.

2.2. Procedure

In the present study a total of six antimicrobial agents

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

(Figure 1).

3. Results

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).

4. Discussion

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.

Antimicrobial

Agent Tested% of Isolates

Inhibited % of Isolates

Inhibited Resistance

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

μg).

High resistance observed for Metronidazole could be

due to the frequent use of the antibiotic in the empiric

treatment of diarrhea [16]. 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%) [18]. 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) [19].

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

K. S. AHMED ET AL.

266

[11]. 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.

5. 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. 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 [20], older agents

such as nitrofurantoin [21] 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.

6. Acknowledgements

We would like to thank our colleagues at our department

for their fruitful discussion during the research and their

technical assistance during manuscript preparation.

REFERENCES

[1] J. D. Penston, “Review Article: Helicobacter pylori Era-

dication under Standable Caution but No Excuse for Iner-

tia,” Alimentary Pharmacology and Therapeutics, Vol. 8,

No. 4, 1994, pp. 369-389.

doi:10.1111/j.1365-2036.1994.tb00304.x

[2] R. J. Laheij, L. G. Rossum, J. B. Jansen, H. Straatman

and A. L. Verbeek, “Evaluation of Treatment Regimens

to Cure Helicobacter pylori Infection—A Meta-Analy-

sis,” Alimentary Pharmacology and Therapeutics, Vol. 13,

No. 7, 1999, pp. 857-864.

doi:10.1046/j.1365-2036.1999.00542.x

[3] F. Mégraud, “Problems Caused by Antibiotic Resistance

of Helicobacter pylori,” La Presse Médicale, Vol. 26, No.

37, 1997, pp. 1775-1780.

[4] D. Y. Graham, “Antibiotic Resistance in Helicobacter

pylori: Implications for Therapy,” Gastroenterology, Vol.

115, No. 5, 1998, pp. 1272-1277.

doi:10.1016/S0016-5085(98)70100-3

[5] J. F. Weel, R. W. van der Hulst, et al., “Heterogeneity in

Susceptibility to Metronidazole among Helicobacter py-

lori Isolates from Patients with Gastritis or Peptic ulcer

Disease,” Journal of Clinical Microbiology, Vol. 34, No.

9, 1996, pp. 2158-2162.

[6] T. Alarcón, D. Domingo and M. López-Brea, “Antibiotic

Resistance Problems with Helicobacter pylori,” Interna-

tional Journal of Antimicrobial Agents, Vol. 12 No. 1,

1999, pp. 19-26. doi:10.1016/S0924-8579(99)00051-5

[7] R. G. Lahaie and C. Gaudreau, “Helicobacter pylori An-

tibiotic Resistance: Trends over Time,” Canadian Journal

of Gastroenterology, Vol. 14, No. 10, 2000, pp. 895-899.

[8] L. C. Mollison, N. Stingemore, et al., “Antibiotic resis-

tance in Helicobacter pylori,” Medical Journal of Austra-

lia, Vol. 173, No. 10, 2000, pp. 521-523.

[9] S. Mendonça, C. Ecclissato, et al., “Prevalence of

Helicobacter pylori Resistance to Metronidazole, Clarith-

romycin, Amoxicillin, Tetracycline, and Furazolidone in

Brazil,” Helicobacter, Vol. 5 No. 2, 2000, pp. 79-83.

doi:10.1046/j.1523-5378.2000.00011.x

[10] H. Miyaji, T. Azuma, et al., “Susceptibility of Helico-

bacter pylori Isolates to Metronidazole, Clarithromycin

and Amoxycillin in Vitro and in Clinical Treatment in

Japan,” Alimentary Pharmacology and Therapeutics, Vol.

11, No. 6, 1997, pp. 1131-1136.

doi:10.1046/j.1365-2036.1997.00258.x

[11] D. K. Bhasin, B. C. Sharma and P. Ray, “Drug Resistance

in Helicobacter pylori Infection,” Indian Journal of Gas-

troenterology, Vol. 19, Suppl. 1, 2000, pp. 29-32.

[12] M. A. Daw, P. Deegan, E. Leen and C. O’Moráin, “Short

Report: The Effect of Omeprazole on Helicobacter pylori

and Associated Gastritis,” Alimentary Pharmacology and

Therapeutics, Vol. 5, No. 4, 1991, pp. 435-439.

doi:10.1111/j.1365-2036.1991.tb00047.x

[13] A. G. Fraser, J. Bickley, R. J. Owen and R. E. Pounder,

“DNA Fingerprints of Helicobacter pylori before and af-

ter Treatment with Omeprazole,” Journal of Clinical Pa-

thology, Vol. 45, No. 12, 1992, pp. 1062-1065.

doi:10.1136/jcp.45.12.1062

[14] R. J. Adamek, S. Suerbaum, et al., “Primary and Ac-

quired Helicobacter pylori Resistance to Clarithromycin,

Metronidazole, and Amoxicillin-Influence on Treatment

Outcome,” American Journal of Gastroenterology, Vol.

93, No. 3, 1998, pp. 386-389.

[15] T. J. Borody, P. Andrews, G. Fracchia, et al., “Omepra-

K. S. AHMED ET AL.

267

zole Enhances Efficacy of Triple Therapy in Eradicating

Helicobacter pylori,” Gut, Vol. 37, No. 4, 1995, pp. 477-

481. doi:10.1136/gut.37.4.477

[16] G. Cederbrant, G. Kahlmeter and A. Ljungh, “The E Test

for Antimicrobial Susceptibility Testing of Helicobacter

pylori,” Journal of Antimicrobial Chemotherapy, Vol. 31,

No. 1, 1993, pp. 65-71. doi:10.1093/jac/31.1.65

[17] M. López-Brea, D. Domingo, I. Sánchez, et al., “Study of

the Combination of Ranitidine Bismuth Citrate and Met-

ronidazole against Metronidazole-Resistant Helicobacter

pylori Clinical Isolates,” Journal of Antimicrobial Che-

motherapy, Vol. 42, No. 3, 1998, pp. 309-314.

doi:10.1093/jac/42.3.309

[18] A. O. Aboderin, et al., “Antibiotic Resistance of Helico-

bacter pylori from Patients in Ile-Ife, South-West, Nige-

ria,” Africa Health Sciences, Vol. 7, No. 3, 2007, pp. 143-

147.

[19] W. D. Chey, et al., “American College of Gastroenterol-

ogy Guideline on the Management of Helicobacter pylori

Infection,” American Journal of Gastroenterology, Vol.

102, 2007, pp. 1808-1825.

doi:10.1111/j.1572-0241.2007.01393.x

[20] G. Dai, N. Cheng, L. Dong, et al., “Bactericidal and Mor-

phological Effects of NE-2001, a Novel Synthetic Agent

Directed against Helicobacter pylori,” Antimicrobial Agents

and Chemotherapy, Vol. 49, 2005, pp. 3468-3473.

doi:10.1128/AAC.49.8.3468-3473.2005

[21] E. M. Quintana-Guzman, M. L. Arias-Echandi, et al.,

“Helicobacter pylori: Susceptibility to Amoxycillin, Ery-

thromycin, Tetracycline, Ciprofloxacin, Nitrofurantoin and

Metronidazole in Costa Rica,” Revista Biomédica, Vol. 9,

1998, pp. 92-96.