Brazilian Journal of Microbiology (2010) 41: 1065-1069

ISSN 1517-8382

ETHAMBUTOL RESISTANCE OF INDIGENOUS MYCOBACTERIUM TUBERCULOSIS ISOLATED FROM HUMAN

Taha Nazir¹, Muhammad Hidayat Rasool², Abdul Hameed³, Bashir Ahmad4, Javed Anver Qureshi5

1
Faculty of Pharmacy, University of Sargodha, Sargodha, Pakistan; 2 College of Pharmacy, Govt. College University, Faisalabad,
Pakistan; 3 Department of Microbiology, Quaid-e-Azam University, Islamabad, Pakistan; 4University College of Pharmacy,
University of the Punjab, Lahore, Pakistan; 5 National Institute of Biotechnology and Genetic Engineering, Faisalabad, Pakistan.

Submitted: October 15, 2009; Returned to authors for corrections: March 03, 2010; Approved: April 26, 2010.

ABSTRACT

The present study was conducted to find out the ethambutol resistance pattern of indigenous isolates of
Mycobacterium tuberculosis from Tuberculosis diagnosed human patients. A total of 172 specimens were
collected from six different sources and comprised of 84.9% sputum, 10.5% pus and 4.7% bronchial
washings. There were 70.9% males and 29.1% females with 84.30% pulmonary and 15.69% extra-
pulmonary tuberculosis. The Mycobacterium tuberculosis isolates collected from primary culture were
further studied to determine their pattern and level of resistance. The inoculums were prepared using 0.5
Mac Farland turbidity standards. Five different concentration of ethambutol were used in Lowenstein
Jensen (LJ) medium i.e. 2µg/ml, 4µg/ml, 6µg/ml, 8µg/ml and 10µg/ml for sensitivity testing. Data showed
10 (5.8%) resistant and 162 (94.2%) sensitive Mycobacterium tuberculosis out of total 172 clinical
isolates. The growth was not inhibited at 1st (2µg/ml) and 2nd (4µg/ml) drug levels, while growth of 50%
isolates inhibited at 3rd level (6µg/ml), 30% inhibited at 4th level (8µg/ml) and 20% at 5th level (10µg/ml).
The last three levels are above the therapeutic index and not recommended in actual clinical practice. It is
thus conceivable to explore some other more effective chemotherapeutic agents, modify combinations or
find more effective procedures to stop morbidity and mortality due to ethambutol resistant Mycobacterium
tuberculosis.

Key words: Ethambutol; Mycobacterium tuberculosis; Resistance; Lowenstein Jensen medium; Human
patients.

INTRODUCTION countries (9) and the disease remained endemic for many
decades (7). Tuberculosis (TB) is the biggest killer of young
Advancements in health care facilities offered a goal to adults in their economically most productive age. Nine million
eradicate tuberculosis (TB) by the end of the 20th century, but it new cases of TB occur every year. Two million people died
reemerged because of the global resistance against anti- annually, that makes it the biggest killer after HIV. Poorly
tubercular drugs. The 95% of TB cases occur in developing implemented directly observed treatment strategy (DOTS)

*Corresponding Author. Mailing address: Assistant professor of microbiology, College of Pharmacy, Govt. College University, Faisalabad, Pakistan.; Tel.:
+92-41-9201036: +92-301-7102378 Fax: +92-41-9201553.; E-mail: drmhrasooluaf@hotmail.com, drmhrasooluaf@gmail.com

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Nazir, T. et al. Ethambutol resistance of M. tuberculosis

gives rise to greater resistance against tubercle bacilli and bronchial washings were collected, labeled and stored,
prevalence of resistant Mycobacteria warrants through DOTS separately. All the samples were centrifuged at 1500 rpm for
plus resistant TB control programs. Almost 38% of the global 15 minutes and decontaminated with NaOH 40g/ L (4%w/v)
TB burden is in the South East Asian (SEA) region where solution. The supernatant fluids were discarded and sediments
annually 7, 50,000 deaths occur due to TB. Five of the 22 high were collected. These concentrated residues of sputum, pus and
burden countries are in the SEA Region (14). bronchial washings were used for primary culture.
Ethambutol act by inhibiting the Biosynthesis of
arabinogalactan. The gene responsible for resistance against Preparation and inoculation of Lowenstein Jensen (LJ)
this drug is EmbCAB that produce the Arabinosyl transferase Medium
that allows continuation of arabinan biosynthesis (12). Lowenstein Jensen medium was prepared by the method
Arabinosyl transferase is an enzyme that is important for the described by Nazir et al. (8) and used for primary culturing of
synthesis of mycobacterial arabinogalactan cell wall. the processed samples. A 15ml of medium was poured into
Resistance is not a serious problem if the drug is employed sterilized 25mL McCartney vials and closed with sterilized
with other antitubercular agents. Ethambutol can be used in silver caps. The medium was autoclaved at 115oC under 15
combination with pyrazinamide, isoniazid and rifampicin. The lb/inch2 pressure for 20 minutes. Then it was solidified in
EmbCAB proteins are believed to be integral membrane slanting position, cooled, labeled and stored at 2-8oC. The LJ
proteins, consistent with their role in the synthesis of various medium slants were inoculated with processed samples in
arabinan-linkage motifs of the arabinogalactan and class-II safety cabinet (Telstar, Spain). The inoculated slants
lipoarabinomannan. Planned and supervised therapy with were kept in incubator at 37oC for 4 weeks. The growth of M.
judicious surgical interference is the only way to ensure cure tuberculosis thus obtained was identified though Acid Fast
for the patient and prevention of the spread of drug resistance staining and further used for sensitivity testing.
(4). Ethambutol is a bacteriostatic drug and specific for most
strains of Mycobacterium tuberculosis and Mycobacterium Preparation of inoculums of Mycobacterium tuberculosis
kansasi. The over seeding of a drug containing medium was
The objective of this investigation was to study the pattern prevented by standardization of inoculums that mitigate the
and level of resistance of indigenous Mycobacterium possibility of miss judgment of considering the resistance to a
tuberculosis isolated from tuberculosis diagnosed human susceptible strain. Approximately 1mg wet weight bacilli/ml
st
patients against ethambutol which is the 1 line antitubercular was estimated to vary between 106 and 108 CFU. The
drug. representative samples of growth containing minimally 50
colonies were taken from primary culture and placed into
MATERIALS AND METHODS McCartney vials containing 1mL of distilled water and 5 glass
beads. The mixtures were homogenized by vigorous stirring by
Collection and Processing of Samples Vortex Mixer (Eyela, Japan) for 1-3 minutes and left in class-II
Pure chemical of ethambutol was obtained from the safety cabinet (Telstar, Spain). The opacity of the suspensions
Schazoo Laboratories (Pvt.), Lahore. A total number of 172 was adjusted by the addition of sterile distilled water to that of
pulmonary and extra-pulmonary tuberculosis diagnosed (AFB a 0.5 Mac Farland turbidity standard. Four serial 10 fold
positive) patients were selected from six different sources. The dilutions of inoculums were prepared i.e. 10-1, 10-2, 10-3 and 10-
4
patients of all age groups were selected, regardless of their age, in sterilized test tubes and labeled as 1, 2, 3 and 4,
gender and previous therapeutic profile. The sputum, pus and respectively. The tube No. 3 of dilution inoculums of 10-3 and

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Nazir, T. et al. Ethambutol resistance of M. tuberculosis

tube No. 5 of dilution inoculums 10-5 were used to culture for 6(3.5%) WAPDA Hospital, Lahore, Pakistan. The specimens
sensitivity testing. comprised of 146(84.9%) sputum, 18(10.5%) pus and 8(4.7%)
bronchial washings with 145(84.30%) from pulmonary and
Susceptibility testing of Mycobacterium tuberculosis 27(15.69%) extra-pulmonary tuberculosis cases. These
The drug sensitivity testing was performed within 1-2 findings are in conformity with Bitar et al. (2) who also
weeks after obtaining primary growth of M. tuberculosis. The reported that majority of cases 89% (n=607) were of
sensitivity was evaluated against ethambutol by drug pulmonary, 6%(n=39) presenting extra-pulmonary tuberculosis
proportion method as recommended by WHO and International and 5%(n=37) cases for whom site of disease was unknown.
Union against Tuberculosis and Lung Diseases (15). The There were 122(70.9%) males and 50 (29.1%) females out
patient’s samples were processed in batches of 10-15 of total 172 clinical isolates. Gender comparison depicts
specimens on drug containing LJ medium and drug free control greater percentage of tuberculosis in males than females. These
LJ medium. Five different concentration of ethambutol were findings have been substantiated by Uplekar et al. (13) who
used in LJ medium i.e. 2µg/ml, 4µg/ml, 6µg/ml, 8µg/ml and reported a seventy percent (70%) excess of males over females
10µg/ml. Each LJ medium slant was inoculated by using the globally each year. The reasons for this difference are unclear
above inoculums prepared from primary cultures of as yet. The findings of this study are also consistent with the
o
Mycobacterium tuberculosis and incubated at 37 C for 4 findings of Haq et al. [3] who also reported 68% male and 32%
weeks. female tuberculosis patients. Our findings are also in
conformity with WHO/ IUALTD (15) which reported 67% of
Recording and interpretation of results male tuberculosis patients.
The Bijoux bottles were inspected weekly for appearance The critical concentration of Ethambutol maintained in LJ
of growth. When the growth was evident on LJ medium, media to consider as border line for declaration of resistant was
colony morphology was noted. One culture bottle was exposed 2µg/ml. The results of this study showed 10 (5.8%) resistant
to day light for one hour and re-incubated to be examined for and 162 (94.2%) sensitive out of total 172 clinical isolates of
pigmentation on the following day. The cultures with no indigenous Mycobacterium tuberculosis. Quantitatively out of
growth were discarded after 8 weeks of incubation. The 10 resistant isolates on LJ medium, 1 (10%) had 10 colonies
presence and amount of growth in term of number of colonies and 9 (90%) had 100 colonies. Our findings are in line with the
on control and drug inoculated medium recorded. The results reports of WHO/ IUALTD, (15) which reported ethambutol
were interpreted for resistance on the basis of percentage of resistance as 5.8% in Iran and 4.1% in china. These finding are
colonies on drug containing media in comparison to the growth also in conformity with Bitar et al. (2) who reported 4.4%
on drug free medium. The strains showing susceptibility were ethambutol resistance. These results are similar to Khan et al.
again incubated and examined at 6 weeks before declaring as (5) who reported markedly increased resistance in Saudi Arabia
sensitive. The growth pattern, number of colonies and during the last 5 years, except pyrazinamide and ethambutol
contamination were checked carefully on weekly basis. showing resistance of 8% & 7%, respectively.
The indigenous Mycobacterium tuberculosis isolates were
RESULTS AND DISCUSSION also inoculated in the medium containing five different
ethambutol concentrations/levels. All of the 10 (100%) isolates
A total number of 172 samples were collected from six were found resistant at 1st (2µg/ml) and 2nd (4µg/ml) levels.
different local sources i.e. 41(23.8%) Outdoor, Mayo Hospital, The 5 (50%) isolates were resistant up to 3rd level (6µg/ml),
110(64%) Indoor, Mayo Hospital, 14(8.1%) Jinnah Hospital, 3(30%) up to 4th level (8µg/ml), and 2 (20%) at higher than 5th

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Nazir, T. et al. Ethambutol resistance of M. tuberculosis

level (>10µg/ml) (Table 1). These concentrations incorporated prevalence of pulmonary tuberculosis than extra-pulmonary
in LJ medium exceed the therapeutic ranges of 3-5µg/ml (1), 2- tuberculosis. Approximately 1/16 of total Mycobacterium
5µg/ml (6) and 4-6µg/ml (11). These exceeding than the tuberculosis isolates were resistant to ethambutol. Maximum
mentioned plasma concentrations may introduce toxicity in resistance was observed at 1st & 2nd drug levels and minimum
actual clinical practice. The most important adverse effects of at above than 5th level. The final concentrations incorporated in
ethambutol are optic neuritis (diminished visual acuity and loss LJ medium have exceeded the therapeutic index, thus not
of ability to discriminate red and green colors), blurred vision, recommended in actual clinical practice. Therefore we have to
and decreased urate excretion which may lead to gout (11). treat resistant patients by replacing and modifying the existing
Additionally drug fever, abdominal pain, headache, dizziness drugs or by exploring some other novel and more effective
and confusion may also occur. Discontinuation of the drug methods to stop the morbidity and mortality due to the
results in reversal of the toxic symptoms (6). ethambutol resistant Mycobacterium tuberculosis.

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