Molecular Characterization of Rifampicin Associated Mutations in Mycobacterium tuberculosis Isolates

Mycobacterium tuberculosis is one of the most lethal pathogens causing infection in 1.8 billion people annually.  Mycobacterium tuberculosis majorly cause pulmonary infection which spreads easily by the air. Rifampicin is a first line drug used for the treatment of this disease. By binding to the 30S ribosomal subunit it inhibits the protein synthesis. The misuse of this drug and incomplete treatment causes alteration at the genetic level of rpoB gene of the MTB. The aim of our study was to find the mutations in the rpoB gene from the clinical isolates of the tuberculosis patients. We collected 412 sputum samples from patients suspected of tuberculosis and cultured in the microbiology laboratory of THQ Fatehpur, Layyah. Positive sputum cultures were analyzed for drug susceptibility test. Genomic DNA was isolated by sonication method using 20 Hz frequency of ultrasonic waves. After that primers were designed using bioinformatics tools for amplification of the gene.  Additionally, the rpoB was amplified by a hemi-nested PCR technique. 91 samples were positive for sputum culture which consisted of 54 males and 37 females. Out of the 91 positive cultures, four samples showed rifampicin resistance. Two samples carried single missense mutation at position 526 and 531 in the amino acid sequence of rpoB gene whereas third and fourth sample carried a single missense mutation at position 516   in the amino acid sequence of rpoB gene. Moreover, 22% of the tested sputum samples were positive for tuberculosis. This means that in the population of tehsil Fatehpur, Layyah tuberculosis is prevalent.  Furthermore, 4.3% of this 22% were found to be rifampicin resistant. In the future researches the harmful effect of rpoB gene mutations which is associated with function of the β-subunit of RNA polymerase in 16S rRNA and its interaction with rifampicin can be estimated by performing molecular docking


Introduction
Mycobacterium tuberculosis (MTB) is the infectious pathogenic agent causing tuberculosis (TB) around the globe. Worldwide, 1.8 billion people are infected by this disease which is approximately equal to one-third of the world population. TB is the foremost cause of deaths globally and it spreads rapidly [1]. When an individual having pulmonary or laryngeal TB sneezes, coughs, laughs and shouts, tiny infectious water droplets are released into the air which infect other healthy individuals. This airborne pathogenic disease is primarily a pulmonary disease but may also affect other organs of the body [2].
The higher concentration of lipid in MTB cells makes them different from other bacteria and resistant to many antibiotics, gives them impermeability to stains and dyes, resistance to osmotic lysis via complement deposition, resistance to toxic oxidations and subsistence inside the macrophages [3]. In the developing countries of the world, malnutrition and TB are the most important problems. Indeed, malnutrition is the predisposed factor which leads a person to secondary immunodeficiency that raises the host's predisposition to the disease. Individuals with TB have a reduced appetite, micronutrient malabsorption, nutrient malabsorption and altered metabolism leading to metabolic wasting syndrome [4]. Another major risk factor for acquiring this disease is the excessive consumption of alcohol [5].
Clinical identification and diagnosis of TB includes the tuberculin or monteux test, sputum culture, chest X-ray and detection by Ziehl-Neelsen method 3 . Sometimes, administering a single drug to the patient leads to the development of resistance in the bacterial population to that drug. The current treatment of TB includes multiple drugs to which organisms are vulnerable.
Administering multiple drugs simultaneously helps in preventing the development of resistance to other drugs. At least 6 to 9 months are required to cure drug-susceptible TB using rifampicinbased treatment [6].
Additionally, at the start of the therapy when the in vitro predisposition of an individual's isolate is unknown, choosing two or more drugs to which the patient's isolate is likely to be susceptible may be tough. So, the improper selection of drugs also leads to the development of drugresistant M. tuberculosis [7]. Mismanagement and misuse of drugs against TB causes resistance in organisms [8], for instance when a patient does not complete their course of treatment, uses poor quality drugs, and suffer from the shortage of drugs and when the doctor prescribes the wrong treatment or duration of time for taking medicines [9].
TB can take the form of multidrugresistant tuberculosis (MDR TB). In this form, MTB is resistant to at least two anti-TB drugs, that is, isoniazid and rifampicin. These drugs are considered as first-line drugs. Another rare type of MDR TB is extensively drug-resistant tuberculosis (XDR TB). This form of TB is resistant to isoniazid and rifampicin as well as to any type of fluroquinolone and also resistant at least to one of the second-line drugs, such as amikacin, kanamycin and capreomycin [10,11]

Study Design and Duration
This experimental study was designed for the identification of TB and of rifampicin mutation in its suspected patients at Tehsil Headquarter Hospital Fatehpur, Punjab, Pakistan. It was carried out at the University Institute of Medical Laboratory Technology (UIMLT), University of Lahore and accomplished at Tehsil Headquarter Hospital Fatehpur, Punjab, Pakistan. The current study took 3 months for completion and was conducted from October 2019 to December 2019.

Sample Size and Collection
Samples were collected from THQ Hospital Fatehpur, Layyah from patients who were suspected of having TB. Samples were cultured in the THQ microbiology laboratory in the biosafety cabinet and were further analyzed for their molecular characterization at the research laboratory of the University of Lahore.
These sputum samples were collected in sterile airtight containers. The collected specimen were labelled with patient name, sex, lab number, date, age, and with other useful information.

Culture and Microscopy of Smear
Sputum samples were cultured on a commercially prepared Lowenstein Jensen (LJ) media for 6 weeks. Positive samples were further analyzed for drug susceptibility tests.
For microscopic detection, the method previously used by Rasol et al. was used with little modification. The smear of the specimen was fixed either by heating or by alcohol fixation on a glass slide and staining was performed with methylene blue. Acid-fast bacillus may appear as pink colouredrod-shaped bacteria whereas the background may stain blue due to methylene blue [20].

Drugs Susceptibility Test
Sputum samples with positive culture were analyzed for rifampicin resistance by performing drug susceptibility tests [21].

Department of Life Sciences
Volume 3 Issue 1, 2021

DNA Isolation
To isolate the DNA by disrupting cells, a sonication method was used. In the sonication procedure known as 8Ml sonication buffer, 50 mM Tris, pH 7.5, and 10 mM EDTA were used with two times centrifugation for 20 mins, one at 2000 × g and other at 2500 × g. The DNA extracted was 50μL [22].

Hemi-Nested PCR
Mutations in the rpoB gene were detected by HN-RT PCR [23]. In this technique, an amplified product is detected with the help of a fluorescent dye. For RNA isolation, master mix was added into the PCR tubes and then these tubes were placed in the thermal cycler for amplification. All chemicals and reagents used for PCR are shown in Table 2. HN-RT PCR program was set with one cycle of polymerase activation at 95°Cfor 5 minutes. It was followed by 30 cycles with denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, elongation at 72°C for 1 minute and final extension at 72°C for 10 minutes. For gene amplification, we used OligoCalc and a specifically designed primer 3 software to design the forward and reverse primer. The forward primer 3' CGATCACACCGCAGACGTTGA and the reverse primer 5' GCCACGCTCACGTGACAGACC 3' were used for amplification.

Statistical Analysis
Data was recorded and compiled in Microsoft Excel. The results were analyzed using the SPSS software (version 22). Frequencies were calculated and the association between gender and age was recorded.

Results
Strains of drug resistant M. tuberculosis (MTB) were grown for 6 weeks using Lowenstein Jensen media under normal conditions in THQ Fatehpur.These strains comprised brown granular colonies. Out of 412 tested sputum samples, only 91(22%) samples tested positive for M. tuberculosis. Out of the total 91 patients tested positive for TB, 54 were male and 37 were female (Figure 1). Only 4 samples were rifampicin resistant, 2 of them were collected from males and 2 from females ( Figure 2). The age wise distribution of all the suspected patients is shown in Figure  3.    In the first positive sample, probe C was mismatched with the rpoB gene due to the mutation occurring at probe C. As a result, TCG changed into TTG, that is, Serine (Ser) switched into Leucine (Leu). In the second sample, probe A was mismatched with the sequence and mutation was present at position 526 in the sequence of probe A. As a result, CAC changed into AAC, that is, Histidine (His) switched into Glycine (Gly) amino acid. The sequence of probe A is (GCACCAGCCAGCTGA). In the third sample, two mutations were detected in probe D and probe E at position 516. Consequently, GAC changed into TAC, that is, Aspartate (Asp) switched into Tyrosine (Tyr). In the fourth sample, a single mutation was found in the region of probe E. Consequently, GAC changed into TAC, that is, Aspartate (Asp) switched into Tyrosine (Tyr). The sequence of probe E is (CGGCTGACAGCCGCGAC) (Figure 4).

Discussion
The However, it is considered as a highly specific and gold standard technique for the analysis of MTB [27]. Multi-drug resistant specimens which showed resistance against rifampicin were further examined for their molecular characterization. The analysis showed different missense mutations in MTB genes [28]. As described earlier, genetic alteration in the rpoB gene is responsible for rifampicin drug resistance. So, our study focused on the gene of interest, that is, the rpoB gene having the size 3519 bp. The area of interest of our gene was 81bp. Its specific region started from codon 506-533, respectively. Two primers were used which were designed using the primer 3 tool. The forward primer (CGATCACAC CGCAGACGTTGA) was positioned at codon 486-493 and the reverse primer (GGCACGCTCACGT GACAGACC) was positioned at codon 537-543, respectively [29]. A previous study showed that worldwide 96.1% of rifampicin resistant specimens have mutations on the rpoB gene. The drug susceptibility test revealed that 61.5% of samples have mutation in the region of the rpoB gene [30]. Another study conducted by Salma Hameed et. al in 2017 justifies our research that showed mutation in rpoB gene. The study revealed that out of 130 multidrug-resistant samples, 61.3% mutations were detected in the rifampicin resistance region (RRDR). At codon number 531, TCG was converted into TTG by the mutation. A single mutation at region 516 converted GAC into TAC and the mutation at position 526 converted CAC into TAC [31]. The mutation of serine to lucien is the most common point mutation reported in rifampicin resistant MTB [32]. The results of our work determined that in the series of mutation frequency, 4 samples of MTB carried a single mutation at locations 516, 526 and 531 respectively on the rpoB gene and this fact is significant for further study on TB in Pakistan

Conclusion
Our study confirmed the existence of mutations in the rpoB gene of MTB in MDR TB patients. A significant percentage (22%) was found to be infected by TB in the population of Tehsil Fatehpur, District Layyah, Punjab. Out of the infected individuals, 4.3% were found to be rifampicin resistant. Moreover, molecular analysis by hemi-nested PCR of the resistant samples showed three-point mutations in the gene and subsequently three missense mutations in the amino acid sequence of the said gene. This result might help others in better understanding M. tuberculosis, the mutations in the rpoB gene and the resistance to the first linedrug rifampicin. In the future, the harmful effect of the rpoB gene mutations associated with the function of the βsubunit of RNA polymerase in 16S rRNA and its interaction with rifampicin can be estimated by performing molecular docking. The docking results may be used further to design better drugs which require less duration for treatment and provide quick diagnostic tools for TB.

• Funding
No funding was received for this study.

• Conflict of interest / Competing interests
The authors declare no conflict of interest.