Isolation-drug resistance profile and molecular characterization of indigenous typical and atypical mycobacteria.

Abstract

One hundred and fifty mycobacterial isolates from different pathological Labs. of Karachi were collected and screened as acid fast. On the bases of phenotypic and biochemical results, it was found that, 58.66% isolates were typical mycobacteria while 41.33% belonged to atypical mycobacteria. The individual percentages of different mycobacterial species include: M. xenopi 35%, M. thermoresistible 19 %, M. terrae complex 6 %, M. marinum 6 %, M. fortuitum 6 %, M. kansasii 25 % and M. tuberculosis 58.66 %. The sensitivity of mycobacterial isolates was determined against 5 first line, 3 second line and 1 third line anti-tuberculosis drugs. The highest number of the isolates (typical and atypical mycobacteria) offered resistance against isoniazid and streptomycin. Clarithromycin was found to be the drug of choice as regards the drug sensitivity in case of atypical mycobacterial isolates. A total of 40 isolates were subjected to PCR based identification and differentiation of 16S rRNA gene(s). Accordingly, 37.5% isolates were identified as typical mycobacteria while 25% were identified as atypical mycobacteria. These findings carry significance because a detailed research based identification (PCR and Multiplex PCR based) regarding indigenous mycobacteria has been reported for the first time in Pakistan. However, both the approaches (conventional and molecular methods) have experimental importance while identifying these organisms.

Full text

Page 1

Pak. J. Pharm. Sci., Vol.24, No.4, October 2011, pp.527-532
527
ISOLATION- DRUG RESISTANCE PROFILE AND MOLECULAR
CHARACTERIZATION OF INDIGENOUS TYPICAL
AND ATYPICAL MYCOBACTERIA

TANVEER KHANUM1, SHEIKH AJAZ RASOOL1*, MUNAZZA AJAZ2 AND ASIF IQBAL KHAN3
1Department of Microbiology, University of Karachi, Pakistan, 2 Federal Urdu University Karachi, Pakistan
3Dow University of Health Sciences, Karachi, Pakistan
ABSTRACT
One hundred and fifty mycobacterial isolates from different pathological Labs. of Karachi were collected and
screened as acid fast. On the bases of phenotypic and biochemical results, it was found that, 58.66% isolates
were typical mycobacteria while 41.33% belonged to atypical mycobacteria. The individual percentages of
different mycobacterial species include: M. xenopi 35%, M. thermoresistible 19 %, M. terrae complex 6 %, M.
marinum 6 %, M. fortuitum 6 %, M. kansasii 25 % and M. tuberculosis 58.66 %. The sensitivity of
mycobacterial isolates was determined against 5 first line, 3 second line and 1 third line anti-tuberculosis drugs.
The highest number of the isolates (typical and atypical mycobacteria) offered resistance against isoniazid and
streptomycin. Clarithromycin was found to be the drug of choice as regards the drug sensitivity in case of
atypical mycobacterial isolates. A total of 40 isolates were subjected to PCR based identification and
differentiation of 16S rRNA gene(s). Accordingly, 37.5% isolates were identified as typical mycobacteria while
25% were identified as atypical mycobacteria. These findings carry significance because a detailed research
based identification (PCR and Multiplex PCR based) regarding indigenous mycobacteria has been reported for
the first time in Pakistan. However, both the approaches (conventional and molecular methods) have
experimental importance while identifying these organisms.

Keywords: Atypical mycobacteria, Typical mycobacteria, Drug sensitivity profile, conventional methods,
Multiplex PCR,


INTRODUCTION

The 22 high tuberculosis burden countries (HBCs)
account for approximately 80% of the estimated new
tuberculosis (TB) cases being reported each year (Floyd et
al., 2002). According to the world health organization
(WHO), nearly 2 billion people (one third of the world's
population) are being exposed to the TB pathogen
annually, 8 million people fall ill, while 2 million people
die from the disease worldwide. Almost 50 % of MDR-
TB (multi drug resistant tuberculosis, defined as resistant
to the two most effective first line anti TB drugs i.e.
rifampicin and isoniazid.) cases worldwide are estimated
to occur in China and India. In 2008, MDR-TB caused an
estimated 150,000 deaths (Falzon et al., 2010). The
problem has further been aggravated by the AIDS factor
(Chum et al., 1996; Mac-Arthur et al., 2001). According
to Gandhi et al (2010), mortality from MDR and XDR-
TB (extensively drug resistant TB defined as MDR-TB
plus resistance to a fluoroquinolone and at least one
second-line injectable agent: amikacin, kanamycin and/or
capreomycin) in their high HIV-prevalence region
(Tugela Ferry, South Africa) is extraordinarily high,
especially during the first month. As in many other
countries, mycobacterium multidrug-resistant strains have
also become a major issue. The incidence of MDR-TB
was found increased in Pakistan from 14% to 47% within
seven years i.e. 1999 to 2006 (Tanveer et al., 2008).
Drug-resistant tuberculosis was first observed in 1948
after the first trials of streptomycin for the treatment of
TB (Robert, 2000). Drug-resistant TB is a public health
issue in many developing countries, as treatment is longer
and requires more expensive drugs. Worldwide
emergence of drug-resistant TB has changed views about
the way we treat infections caused by Mycobacterium
tuberculosis (M. tuberculosis). This change reflects our
understanding of the failures of standard regimens in
patients suffering from drug-resistant strains (Becerra et
al., 2000; Espinal et al., 2000). Atypical mycobacteria
cause neither TB nor leprosy, but they do cause
pulmonary diseases resembling TB (Van Crevel et al.,
2001). The distribution and the incidence of disease
caused by them are not fully understood in most parts of
the world. These organisms are widely distributed in
nature (Kazda, 1983) and have been isolated from natural
water, tap water, soil water used in showers and surgical
solutions. In United States, most of the isolates reported
include M. avium, M. kansasii and M. fortuitum (O’ Brien
et al., 1987). There have been some reports of atypical
mycobacteria from Japan (Tsukamura et al., 1988). In
most of the studies from India M. tuberculosis has been
found as major cause of mycobacterial infections and the
proportion of atypical mycobacteria has been considered
low. Thus, species like M. fortuitum, M. avium and M .
scrofulaceum have reportedly been isolated by Sachdev et
al. (2002). Stratmann et al. (2002) had used polymerase
chain reaction (PCR) techniques for the detection and
rapid identification of the clinically relevant mycobacteria
*Corresponding author: e-mail: rasoolajaz@yahoo.com

Page 2

Isolation-drug resistance profile
Pak. J. Pharm. Sci., Vol.24, No.4, October 2011, pp.527-532
528
using methods for concentration and detection of M.
avium, M. intracellulare from the clinical specimens and
M. paratuberculosis from clinical specimens and milk.
Roth et al (2000) reported PCR assays using genus/ group
specific amplification followed by restriction analysis for
the detection of gene regions like 65 KDa and rRNA gene
region which have been found useful for identification of
different mycobacteria. Ruiz et al. (2002) declared the
merits of PCR amplification followed by hybridization for
the detection of M. avium, M. chelonae, M. scrofulaceum,
M. ulcerans and other mycobacteria.

MATERIALS AND METHODS

A total of 150 (including typical and atypical)
mycobacterial isolates were procured from different
pathological labs of Karachi (table 1). Phenotypic
identification of typical and atypical mycobacteria was
performed by different methods as described in ASM
manual (Pfyffer et al., 2003) that included rate of growth,
pigment production, colonial morphology and growth at
different temperatures (37oC, 45oC and room temperature
i.e., 20-25oC) while the selection of specific biochemical
tests for identification was made on individual basis after
the growth rate, pigment production and colonial
morphological features were noted as described by
Niemann et al. (2000). In addition, tests based on Tween-
80 hydrolysis, nitrate reduction , urease production, niacin
accumulation, heat stable catalase and semi quantitative
catalase production, growth
agar(without crystal violet), and pyrazinamidase
production were done (Kent and Kubica, 1985). Drug
resistance profile was done by resistance ratio method in
which different drugs were selected for susceptibility
testing (Acharya et al., 2008). Serial two fold dilutions of
the drugs, first line (isoniazid, rifampicin, streptomycin,
ethambutole and pyrazinamide)
(ciprofloxacin, amikacin and sparfloxacin) third line
(clarithromycin) were made in Lowenstein Jensen (LJ)
medium. Molecular based
differentiation of typical and atypical mycobacteria were
done by EZTBPCR kit from MBDr Diagnostics (Bio
Diagnostic Research Company, Malaysia). Thermo
stabilized PCR mix with specific primers was used for the
detection of typical and atypical mycobacteria However,
multiplex-polymerase chain reaction was also used by
Kapur et al. (1995). As described by Gopinath and Singh
(2009). Thus, a number of amplified products were
obtained. All the protocols were followed according to the
manufacturer’s recommendations. In short, for PCR, 15µl
of DNAse RNAse free water was added to each thermo
stabilized PCR mix tube. Extracted DNA sample (5 µl
template DNA) was added to the thermostable PCR mix.
Positive & negative controls were also run. Tubes were
placed in PCR machine (Thermo Electron Corporation,
USA). Results were analyzed after gel electrophoresis of
the amplified DNA; TB DNA ladder and 100 bp DNA
on MacConkey’s
second line
identification and
ladder (Promega; 0.13µg µl-1) were used for comparison
of the results (Sambrook et al., 1989).

RESULTS

On the bases of phenotypic and biochemical results it
was concluded that out of one hundred and fifty isolates,
58.66 % were typical mycobacteria while 41.33 % were
atypical mycobacteria. The individual percentages of
different mycobacterial species include: M. xenopi - 35%;
M. thermoresistible – 19 %; M. terrae complex – 6 %; M.
marinum – 6 %; M. fortuitum – 6 %; M. kansasii – 25 %
and M. tuberculosis- 58.66 % (fig. 1). According to
resistance ratio method (table 2) rifampicin resistance was
offered by 6.2 % and 9.68 % of typical and atypical
respectively, while 93.8 % and 90.32 % were found
sensitive respectively. Isoniazid resistance (26 % isolates)
was shown by typical while 59.9 % resistant isolates
belonged to atypical mycobacteria while sensitivity was
offered by 74 % and 40.1% isolates respectively.
Similarly resistance against streptomycin was shown by
22 % (typical) and 16.9 % (atypical) isolates while
sensitivity was offered by 78% and 83.1% of the isolates
respectively. Ethambutol resistance was offered by 14.2
% and 15.9 % of the typical and atypical mycobacterial
isolates (respectively), 85.8 % and 84.1% of the isolates
were found sensitive against ethambutol. Resistance
against pyrazinamide was offered by 32.9 % of typical
and atypical mycobacterial isolates while equal number
(67.1 %) of both the types were found sensitive.
Multidrug resistance was offered by 6 % and 2 % of the
typical and atypical isolates respectively. Over all highest
resistance (32.9 % by both the types) was offered against
pyrazinamide, followed by isoniazid resistance by typical
26 % and 59.9 % by atypical, 22 % and 16.9 % for
streptomycin, 14.2% and 15.9% for ethambutol and 6.2 %
and 9.68% for rifampicin (for both typical and atypical
mycobacterial isolates respectively). According to our
findings, the most suitable drug to control both typical
and atypical mycobacteria is rifampicin (fig. 2).
Although, the atypical isolates were found more resistant
to isoniazid and rifampicin however, the frequency of
MDR was low among the atypical (fig. 2). In case of
second line drugs, the highest number of isolates(typical
mycobacteria) offered resistance against ciprofloxacin
while the atypical offered highest resistance against
amikacin. The second line drug of choice to control (as
per in vitro investigations) TB infection by atypical
mycobacteria has been found to be sparfloxacin, while
clarithromycin (third line) can be rated as the drug of
choice for both the mycobacterial types (fig. 3). On the
basis of genotypic results, 37.5 % and 25 % of the isolates
were identified as typical and atypical mycobacteria
respectively. Figure 4 presents the DNA bands of
different isolates (sized using Mycobacterium DNA
ladder). Lane wise description of the bands is given in fig.
4.

Page 3

Tanveer Khanum et al.

Pak. J. Pharm. Sci., Vol.24, No.4, October 2011, pp.527-532
529
Table 1: Procurement of the isolates from different pathological labs of Karachi

Labs and hospitals Essa’s lab
10
50
SIUT
100
0
Ehsan Ullah’s Lab
20
0
OICD
20
0
No of collected isolates
No of collected biological sample

Key: NCI - No. of collected isolates, NCB - No. of collected biological sample, ESSA’S LAB - Dr. Essa’s Laboratories, SIUT -
Sindh Institute of Urology and Transplantation, Ehsan Ullah’s Lab - Dr. Ehsanullah Laboratories, OICD - Ojha Institute of Chest
Diseases.

Table 2: Drug (first line) resistance profile of typical and atypical mycobacteria

Typical mycobacteria
R(%) S (%)
1 Rifampicin 6.2 93.8
2 Isoniazid 26 74
3 Streptomycin 22 78
4 Ethambutol 14.2 85.8
5 Pyrazinamide 32.9 67.1

Key: S - sensitive, R - resistance. The results are based on the resistance ratio method.

S. No. Drugs
Atypical mycobacteria
R (%) S (%)
9.68 90.32
59.9
16.9
15.9
32.9
MDR (%) MDR (%)
6 (5.68) 2 (3.2)
40.1
83.1
84.1
67.1







Fig. 1: Identification of mycobacteria up to species level.


Fig. 2: Comparison of first line drug resistance pattern of typical and atypical mycobacteria.
Key: R=Rifampicin, I=Isoniazid, S=Streptomycin, E=Ethambutol, PZA= Pyrazinamide

Page 4

Isolation-drug resistance profile
Pak. J. Pharm. Sci., Vol.24, No.4, October 2011, pp.527-532
530
DISCUSSION

Nontuberculous
opportunistic pathogens frequently found in water
sources, soil, dust, and air. Infections from animals i.e.
cattles (Bollo et al., 1998), dogs (Wallace, 1989) and cats
(Ngan et al., 2005) have been described. These organisms
are implicated in infections of the skin, bones and soft
or atypical mycobacteria are
tissues.
susceptible to the disseminated infection caused by
nontuberculous mycobacteria (Porat & Austin, 2008;
Prendiki et al., 2008). Although, diseases caused by these
organisms are uncommon (compared with the classical
tuberculosis) but a significant increase in pulmonary and
non pulmonary infections by mycobacteria has been
observed during the last two to three decades (Wolinsky
Immunocompromised patients are most


Fig. 3: Comparison of second and third line drug resistance pattern of typical and atypical mycobacteria.
Key: Cf=Ciprofloxacin, Amk=Amikacin, Spr =Sparfloxacin, *Cla=Clarithromycin (A third line drug)

Test 1 Test 2 Test3 Test 4 Test 5 Test 6 Test 7 Test 8

Fig. 4: PCR based differentiation of typical and atypical mycobacteria
Key: Lane 1 and 12: MTB DNA ladders.
Lane No.2: positive control (showed 541, 383, 211 and 127 bp bands).
Lane No.3: negative control (showed only 663 bp band, PCR control.
Lane Nos. 5, 10, 11 are indicative of M .tuberculosis as they showed all 541, 383, 211 and 127 bp bands; lane No.4 and 6 showed
DNA products that gave bands at 663 and 383 bp positions which are specific for atypical mycobacteria.

Page 5

Tanveer Khanum et al.

1979; Wayne and Sramek 1992). Our results show that
the frequency of atypical is lower compared to the typical
mycobacteria and the results are in agreement with the
findings of Agarwal (2001). However, the prevalence of
atypical mycobacteria varies from country to country and
region to region. Mawak et al. (2006) reported 61.54 %
M.tuberculosis, 15.38 % M. bovis and 23.08% as atypical
mycobacteria. Among them 20.69 % were classified as
M. avium while M. kansasii and M. fortuitum percentage
was found to be the same i.e. 3.45%. Reports are available
that the drug susceptibility profile of atypical is usually
quite different from typical mycobacteria. Firstly, these
organisms are frequently sensitive at high concentrations
of antimycobacterial drugs (Wallace et al., 1990). These
results are also in agreement with the ones reported by
Katoch and Mohan (2001); according to them first, higher
cut off values for deciding sensitivity or resistance are
needed. Secondly, rapidly growing mycobacteria are
usually resistant to rifampicin and isoniazid, whereas
these are found sensitive to drugs like new generation
macrolides, cephalosporins and sulphones (Katoch, 2004).
These observations are partially in agreement as we have
reported amikacin resistant isolates (figure 3). Javaid et
al. (2008) reported the prevalence of MDR among
untreated patients in NWFP as 2.5 %, whereas we found 6
% and 2 % for typical and atypical mycobacterial isolates
respectively which is a major cause of concern and should
be addressed through effective TB control programme,
preferably with DOTS strategy. The multiplex PCR
method could not clearly differentiate M. tuberculosis
complex and NTM strains. In addition, restriction
fragment length polymorphism analysis and direct
sequencing of the amplicon of NTM could be used to
supplement species identification. Moreover, the standard
culture method (considered a gold standard) proved to be
100-fold more sensitive than the EZTBPCR Test Kit.

ACKNOWLEDGMENT

The authors wish to acknowledge the valuable
cooperation of Dr Rafiq Khanani (Director of the
Research Laboratories, Dow University of Health
Sciences, Karachi) for extending the laboratory facilities
to carry out a part of this research work.

REFERENCES

Acharya S, Ghimire P, Khadka DK and Nepali S
(2008).Comparison of Proportion and Resistance Ratio
Methods for Drug Susceptibility
Mycobacterium tuberculosis Isolated from Patients
Attending National Tuberculosis Centre, Nepal. SAARC
J. Tuber. Lung Dis. HIV/AIDS, 5(1): 13-20.
Agarwal NJ (2001). Isolation rates of non-tuberculous
mycobacteria from Amritsar Indian. J. Med. Microbiol.,
19(4): 230-231.
Pak. J. Pharm. Sci., Vol.24, No.4, October 2011, pp.527-532
531
Testing of
Becerra MC, Freeman J, Bayona J, Shin SS, Furin JJ,
Kim JY, Werner B, Timperi R, Sloutsky A, Wilson
ME, Pagano M, Farmer PE (2000). Using treatment
failure under effective directly observed short-course
chemotherapy programs to identify patients with
multidrug-resistant tuberculosis. Int. J. Tuberc. Lung
Dis., 4: 108-114.
Bollo E, Guarda F, Capucchio MT and Galiett F (1998).
Direct Detection of Mycobacterium tuberculosis
complex and M. avium complex in tissue specimens
from cattle through identification of specific rRNA
sequences. J. Vet. Med.,Series B. 45: 395-400.
Chum HJ, O'Brien RJ, Chonde TM, Graf P, Rieder HL.
(1996). An epidemiological study of tuberculosis and HIV
infection in Tanzania, 1991-1993. AIDS, 10: 299-309.
Espinal MA, Kim SJ, Suarez PG, Kam KM, Khomenko
AG, Migliori GB, Baéz J, Kochi A, Dye C, Raviglione
MC (2000). Standard short-course chemotherapy for
drug-resistant tuberculosis: treatment outcomes in 6
countries. JAMA, 283: 2537–2545.
Floyd K, Blanc K, Raviglione M and Jong-Wook L
(2002). Resources required for global tuberculosis
control. Science. 295 (5562): 2040-2041
Falzon D, Glaziou P, Jaramillo E, Mirzayev F, Nathanson
E, Sismanidis C, Gemert W, Weil D and Zignol M.
(2010). Multidrug and extensively drug-resistant TB
(MDR/XDR). 2010 global report on surveillance and
response. World Health Organization., pp.1.
Gopinath K and Singh S (2009). Multiplex PCR assay for
simultaneous detection
Mycobacterium tuberculosis, Mycobacterium avium
complexes and other Mycobacterial species directly
from clinical specimens. J. Appl Microbiol., 107: 425-
435.
Gandhi NR, Shah NS, Andrews JR, Vella V, Moll AP,
Scott M, Weissman D, Marra C, Lalloo UG, Friedland
GH (2010). HIV coinfection in multidrug- and
extensively drug-resistant tuberculosis results in high
early mortality. Am J. Respir. Crit. Care Med., 181: 80-
86.
Kent PT, Kubica GP
Mycobacteriology : a guide for the level III laboratory.
US Department of health and human services,
Publication no. (CDC) 86–8230 Center for Disease
Control., Atlanta.
Kapur V, Li LL, Hamrick MR, Plikaytis BB, Shinnick
TM, Telenti A, Jacobs WR, Banerjee A, Cole ST,
Yuen KY, Clarridge JE, Kreiswirth BN and Musser JM
(1995). Rapid species assignment and unambiguous
identification of mutations
antimicrobial resistance in Mycobacterium tuberculosis
by automated DNA sequencing. Arch. Pathol. Lab.
Med., 119: 131-138.
Kazda JF(1983). The principles of ecology of
mycobacteria. In: Biology of Mycobacteria, Stanford
JL, Ratledge C, (editors) Vol.2. Academic Press,
London, 323-342.
and differentiation of
(1985) Public health
associated with