Molecular detection and characterization of resistant genes in Mycobacterium tuberculosis complex from DNA isolated from tuberculosis patients in the Eastern Cape province South Africa
© Bhembe et al.; licensee BioMed Central Ltd. 2014
Received: 30 April 2014
Accepted: 1 September 2014
Published: 4 September 2014
Tuberculosis (TB) in both animals and humans is caused by Mycobacterium tuberculosis complex (MTBC) primarily transmitted by inhalation of aerosolized droplets containing the organism. Multi-drug resistance (MDR) and extensive drug resistance (XDR) are evolutionary features of Mycobacterium tuberculosis to subvert the antibiotic regimes in place. The heavy burden of TB worsened by HIV endemic in South Africa motivated for the investigation of MTBC prevalence among TB patients in Port Elizabeth and the amplification and sequencing of the DNA amplicons known to confer resistance to TB drugs.
Three thousand eight hundred and ten (3810) sputum specimens were processed and DNA was isolated from sputum specimens collected from different hospitals and health care places in the Eastern Cape Province, South Africa. DNA was amplified using the Seeplex® MTB Nested ACE detection assay. The agar-dilution proportion method was used to perform drug-sensitivity testing using 7H10 Middlebrook medium. Target genes known to confer resistance to first and second-line drugs were amplified and the amplicons sequenced.
One hundred and ninety (5%) DNA samples tested positive for MTBC and from the resistant profiles of the 190 positive samples, we noted that multidrug-resistant TB was identified in 189 (99.5%) with 190 (100%) patients infected with MTB resistant to isoniazid and 189 (99.5%) having MTB resistant to rifampicin. Other percentages of drug resistance observed including 40% pre-XDR and 60% of XDR.
This study provides valuable data on the different kinds of mutations occurring at various target loci in resistant MTBC strains isolated from samples obtained from the Eastern Cape Province. The results obtained reveal a high incidence of MDR amongst the positive samples from Eastern Cape Province, South Africa.
KeywordsMycobacterium tuberculosis complex Tuberculosis Multidrug resistance Extensive drug resistance
The Mycobacterium tuberculosis Complex (MTBC) consists of nine bacterial species that cause tuberculosis (TB) in mammals, including human beings . MTBC results in substantial economic losses in cattle herds and humans as it is usually found in the more economically active humans . Tuberculosis is a major public health concern and a third of the world’s population is infected with some members of MTBC . South Africa is a country with high incidence of TB, there were 550 cases per 100 000 population in 2003, 718 case per 100 000 population in 2004 [3, 4] and 600 cases per 100 000 population in 2005 . The country had one of the worst recorded epidemics in the world in 2008 caused by the rising rates of HIV and the emergence of multidrug resistant TB . The country is divided into nine provinces and among them the Eastern Cape has 80% of TB cases in South Africa . The Eastern Cape is one of the poorest Provinces in South Africa and because of its poverty the spread of TB is enhanced . Most people take TB as a disease of the past decade caused by strains that cannot be treated with existing drugs; this disease has turned to be one of the world’s most pressing health problems .
Resistance of the organisms to TB drugs is a major public health problem that threatens the progress made in TB control worldwide. Drug resistance arises due to improper use of antibiotics in chemotherapy of drug susceptible organisms . Multidrug-resistance TB (MDR-TB) is resistant to the two most commonly used drugs (isoniazid and rifampicin) in the common four drug regimen . In 2010, the World Health Organization (WHO) estimated that there were globally 290 000 cases of MDR-TB among cases of pulmonary TB that were reported . There have been 1.8% increases in MDR-TB cases in South Africa. There are several factors that contribute to the development of MDR-TB, such as poor adherence of patients to first line anti-TB drugs, dosage and duration of treatment, inappropriate treatment regiments and non-compliance to national guidelines and TB protocol by TB clinicians .
Inappropriate use of second line drugs used in the treatment of TB leads to amplification of resistance and development of XDR-TB . Extensively drug resistance TB (XDR-TB) is the TB resistant to any fluoroquinolone and at least one of the injectable drugs (capreomycin, kanamycin and amikacin) in addition to isoniazid and rifampicin. Several methods have been used to identify MTBC, including culture and biochemical tests such as acid-fast smears and sputum cultures. The diagnosis of TB includes history, physical examination and radiological findings in lung apices. Acid-fast smears and cultures of sputum are also required . This study aimed at using a multiplex PCR targeting two genes (mpb64 coding for immunogenic secretory protein specific for Mycobacterium tuberculosis complex and IS6110 insertion element found exclusively within the members of the MTBC) for the detection of MTBC in sputum samples obtained from Eastern Cape Province and determination drug resistance using sequencing of the DNA amplicons known to confer resistance to TB drugs.
Three thousand eight hundred and ten sputum specimens in the Eastern Cape Province were collected from different hospitals and clinics from patients that showed clinical signs of TB and transported to the microbiology laboratory at Fort Hare University, South Africa for culture over a period of 24 months from January 2012 to 2013 December. Biodata including age (0–20; 21–40; 41–60 and 60 years and above) and gender of the patients were also collected.
The bacteriological procedure was done as outlined by Balows et al. . NaOH (4%) was used to kill any other contaminants in this procedure. Two Lowenstein-Jensen (LJ) slants were inoculated and incubated at 37°C for 6–8 weeks. A smear was prepared from each of the processed samples on a grease-free slide and stained by carbol fuschin using the Ziehl_Neelsen technique. Slides were checked for AFB under a microscope. Mycobateria were isolated from sputum. Isolation and identification of mycobacteria was carried out by the Microbiology service of each hospital using acid-fast staining (AFB).
Antibiotic susceptibility profiles were done at the National Health Laboratory Services in Port Elizabeth. Several spade-full of growth were scraped from LJ slants, transferred to a sterile screw-cap tube containing glass beads and 3 ml normal saline (0.85%) and mixed well on a vortex mixer. Turbidity was matched against McFarland standard no. 1. Inoculum (100 μl) was added to each plate, containing 5 ml 7H10 Middlebrook medium with drug in each quadrant. M. tuberculosis strain H37Rv was used as control in all sets of experiments. The inoculated plates were incubated at 37°C in an atmosphere of 10% CO2.
The agar-dilution proportion method was performed according to Balows et al. . Lyophilized drugs were reconstituted aseptically in water. The stock was diluted in such a manner that a 5 μl aliquot contained the requisite amount of each drug. The drug concentrations used in this study were isoniazid (1 μg ml-1) Rifampicin (5 μg ml-1), Streptomycin (10 μg ml-1), Ethambutol (10 μg ml-1), Ethionamide (5 μg ml-1), ofloxacin (2 μg ml-1), Amikacin (6 μg ml-1) and capreomycin (40 μg ml-1). Results were recorded after 3 weeks. Each drug-sensitivity test was carried out at least three times and the average was recorded.
Identification of MTBC species using Seeplex®MTB nested ACE detection assay
The Seeplex® MTB Nested ACE detection assay (Seegene Inc, Korea) was carried according to the manufacturer’s instructions using a thermal cycler (Bio-Rad, South Africa). The assay is a multiplex PCR involving the first PCR (1 cycle at 94°C for 15 min; 40 cycles at 94°C for 30 s, 60°C for 30 s, 72°C for 30 s; 1 cycle at 72°C for 5 min) and a nested PCR (1 cycle at 94°C for 15 min; 30 cycles at 94°C for 30 s, 62°C for 30 s, 72°C for 30 s; 1 cycle at 72°C for 5 min). The amplicons were separated on 2% agarose gel electrophoresis, at 100 V for 90 minutes using TBE buffer pH 8.3. The gel was thereafter visualized under Alliance 4.7 transilluminator (UVITEC Limited, Cambridge, UK).
Kat G, rpoB , rrs and eis gene amplification through Polymerase Chain Reaction
Resistant genes to first-line drugs were amplified using primers RTB511f (5′ TGGCACGCTGCCGGCACCTA) and RTB 311r (5′ CGAAGCCGAACCCGAACGTC) for katG gene. After initial denaturing at 93°C for 5 min, 3-step cycling for 30 amplification cycles were completed each consisting of 1 min at 95°C, 1 min at 64°C and 2 min at 72°C. A final extension of 10 min at 72°C was applied. For the rpoB gene the RDRSf (5′GTCGGTCATGTTCGCGATCG) and RDRAr (5′ TCGGCCAGGTAGTCGCTGAT) primers were used. After initial denaturing at 95°C for 5 min, 3-step cycling for 40 amplification cycles were completed each consisting of 1 min at 95°C, 1 min at 64°C and 1 min at 72°C. A final extension of 10 min at 72°C was applied. The molecular detection of resistant genes to second line drugs was done by amplifying the DNA with second line drugs primers; RRS2f (5′ TGCCGGGGTCAACTCGGAGG) and RRS2r (5′ GAACCCCTCACGGCCTACGC) for the rrs gene. After initial denaturing at 94°C for 4 min, 3-step cycling for 35 amplification cycles were completed each consisting of 1 min at 94°C, 1 min at 58°C and 2 min and 30 s at 72°C. A final extension of 10 min at 72°C was applied. Eisf1 (5′ GCCATGGGACCGGTACTTGC) and Eisr1 (5′ GTAGATGCCGCCCTCGCTAG) for the eis gene was the second primer used to amplify resistant genes to second line drugs with initial denaturing at 94°C for 4 min, 3-step cycling for 35 amplification cycles were completed each consisting of 1 min at 94°C, 1 min at 54°C and 2 min and 30 s at 72°C and a final extension of 10 min at 72°C.
The samples were amplified by PCR using the synthetic oligonucleotide primers that have been mentioned above. PCR was carried out in 25 μl tube containing 12.5 μl of master mix with (Tris pH 8.0, MgCl2, dNTP, 1UTaq polymerase), 8.5 μl water (DDW molecular grade), 1 μl of each primer and 2 μl of DNA template . Mycobacterium H37RV strain was used as a positive control and sterile water used as a negative control. The amplified products were separated in 2% agarose gels together with 100 bp ladder (Fermentas), applied in a separate lane. Gels were stained with ethidium bromide and photographed on UV transiluminator.
Sequencing of KatG, rpoB, rrs and eisgenes
To check for resistance and possible mutations, twelve amplicons per gene of the amplified products were sequenced. DNA sequencing was performed using a Big Dye Terminator DNA sequencing kit v3.1 (Applied Biosystems, UK). Direct sequencing was done with 2 μL of chromosomal DNA, 0.25 μL of primer (10 pmol per μL), 2 μL of Big Dye buffer and 2 μL of Big Dye. Cycle parameters included a denaturation at 96°C for 10 s, annealing at 50°C for 20 s, and extension at 60°C for 4 min over 30 cycles, followed by Agencourt CleanSeq clean up. Sequences were determined by electrophoresis with the ABI 3130xl DNA sequencer (Applied Biosystems, UK). Editing of the sequences was performed using Bioedit Alignment Editor. Cleaned sequences were sent to BLAST using the nBLAST in NCBI (http://www.ncbi.nlm.nih.gov/); resistant genes were categorized to resistance types by their resistance profiles and sequence similarity . The study was approved by the research ethics committee of the Goven Mbeki Research and Development Centre, University of Fort Hare, Record Number 2012/2013-01356. Written informed consent from participants was obtained and the confidentiality of the patients’ identities was protected.
Results and discussion
Amplification of Seeplex DNA samples results
Males N (%)
Females N (%)
Blacks N (%)
Mixed race N (%)
Whites N (%)
When looking at the statistics by Census , black Africans are more than any other race in this Municipality. This results show high prevalence of MTBC amongst the black race which could be due to the fact that these samples were collected from public clinics, where most black people frequently visit because of the low cost. Some mixed race people attend public clinics and some attend private clinics and that could be one of the reasons there was 15.8% mixed race that had MTBC. However it could also be because of sampling bias where only samples from eastern part of the Eastern Cape were received irrespective of gender or race. The mixed race and black people work together most of the time and some attend the same schools and stay in the same location which increases the risk of transmission of MTBC between the two races.
A fascinating observation from our results (Table 1) was made in this study where were observed that there are more females (56.8%) in this study that were detected to have MTBC in comparison to males (43.16%) which contradicts what other studies have reported on [6, 17]. It has been reported that in most of the world, more men than women are infected by MTBC . A report by Census  gave the population of the people in Nelson Mandela Bay Metropolitan Municipality 552 994 (48%) are male and 599 121 (52%) are female which shows that there are more females than males in this region. However, our results concerning females being more infected by MTBC compared to males might be due to the fact that females care for the sick, both children and their husbands or brothers. It can also be as a result of selection bias where more samples for females were chosen than males.
Nevertheless most women die due to TB  and this affect woman mainly in their economically and reproductively active years . This was shown in our study (Table 1) where 47% of women aged 15–44 years are the most infected. Our results are also in agreement with a study by Murray et al.  who found out that in women aged 15–44 years in developing countries TB is the most common cause of morbidity and mortality combined, and it kills more women than any other infectious disease including malaria and AIDS . It was noted that the group from 15–44 years is a sexually and economically active age groups which could be another reason of having more females detected with MTBC. This does not mean that TB is contracted through sexual intercourse but it is transmitted by having a close contact with someone infected or an animal. This result (Table 1) indicates that TB can infect any race exposed to MTBC despite their genetic make-up and age group.
Drug susceptibility results
One hundred and ninety (100%) of the patients were found to be resistant to at least one or more anti-TB drugs (Table 1). Resistance to only one drug was found in all 190 patients who were infected with MTBC resistant to isoniazid 190 (100%) as the highest resistance while the lowest resistant profile was observed on ethambutol with 9.5%. Almost all the samples (99.5%) that were resistant to isoniazid were also resistant to rifampicin. The findings of the study supports what other authors hypothesized which states that rifampicin can be used as a surrogate marker for MDR, this is due to the fact that 99.5% of rifampicin resistant Mycobacterium tuberculosis strains are equal to isoniazid [19–21]. Only one (0.53%) sample was susceptible to rifampicin. This is a first report of high drug resistant MTBC Port Elizabeth; these results are higher than those that were reported by Green et al.  who reported on 58.4% MDR-TB in the Mpumalanga Province of South Africa.
Susceptibility and resistance profile of M. tuberculosis genotypes to first and second line anti-mycobacterium drugs
TB resistance profiles to first line (FL) drugs
TB resistance profiles to second line (SL) drugs
On the second line drugs resistance to capreomycin was the lowest 13 (6.8%) and the highest was amykacin in 83 (43.7%). Pre-extensively drug tuberculosis was detected in 33 (17.4%) patients and extensively drug resistant TB was detected in 157 patients. Thirty three isolates (17.4%) were identified as pre-XDR and one hundred and fifty seven isolates were identified as XDR-TB. Comparing our results with the study by Campbell et al.  who reported ofloxacin (69: 21.9%) and amykacin (1:0.3%), our values are only high in amikacin resistance which was 43.7%. Of the tested isolates 55 (16%) were susceptible to all the study antibiotics and 10 (3%) were determined to be XDR- M. tuberculosis. Our results show a high drug resistance of second line drugs in the Eastern Cape, South Africa.
Mutation conferring resistance to INH, RIF, CAP and KAN drugs
Frequency of mutations in katG gene codons 293, 315 and 463 in 140 INH-resistant strains of M. tuberculosis complex
katGgene mutation positions
N293 → G
S293 → G
S315 → T
S315 → N
T315 → N
T315 → S
R 463 → L
No. of strains (%)
Frequency of mutations in rpoB gene codons 42, 52, 87, 92, 441, 450 and 457 in 140 RIF-resistant strains of M. tuberculosis complex
rpoBgene mutation positions
Y42 → D
G52 → A
H87 → G
L92 → S
V441 → G
L450 → S
L 457 → P
No. of strains (%)
The rrs gene it consist of injectable anti-TB drugs such as amikacin (AMK), kanamycin (KAN) and capreomycin (CAP) . Proper use of injectable drugs is critical to the effective treatment of MDR-TB and in prevention of XDR-TB . Mycobacterium culture and susceptible testing in media either solid or liquid relies on conventional diagnosis of MTBC strains . This method is not reliable for the detection of injectable drugs resistance . AMK and KAN bind to the 16S rRNA in the 30S ribosomal subunit and inhibit protein synthesis  and CAP interferes with translation and inhibits phenylalanine synthesis in mycobacterial ribosome . Mutations in the MTBC that prevents the binding of the injectable drugs to the targeted pathogen gene have been associated with resistance to the three injectable drugs [52, 53].
Frequency of mutations in rrs gene showing nucleotide change in 120 rrs -resistant strains of M. tuberculosis complex
rrsgene mutation positions
S2169,70 → A
R2201 → R
K2202 → E
No. of strains (%)
The results obtained from this study show a high prevalence of MTBC among Eastern Cape population. Of noteworthy is the fact that women at their reproductive years are mostly infected and this could lead to a vicious cycle, hence women are exposed to a lot of people. The study also revealed a high prevalence of MDR amongst the Eastern Cape population.
Acquired Immunodeficiency Syndrome
Drug Susceptibility Testing
human Immune Virus
Mycobacterium Tuberculosis Complex
Polymerase Chain Reaction
the gene that encodes the β subunit of bacterial RNA polymerase
World Health Organization
Extensively Drug Resistant
The authors acknowledge the National Research Foundation (NRF) and Govan Mbeki Research and Development Centre (GMRDC), University of Fort Hare for financial support. We also wish to acknowledge the staff at the NHLS TB referral lab in Port Elizaberth where the samples were collected.
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