Evaluation the Expression of Eux Pump Genes “Tap & P55 in Mycobacterium Tuberculosis Clinical Isolates in Fars, Iran

The increasing drug resistance in Mycobacterium tuberculosis isolates has become a global problem for tuberculosis therapy programs. Genetic mutations in rifampin (RIF), one of the key drugs in the treatment of tuberculosis are main mechanism of resistant to this drug in M. tuberculosis. Absence of mutation in target genes, other mechanisms such as eux pump suggests possible role of drug resistant. The objective of this study was to nd out mutations in rpoB genes in rifampin resistant isolates and to compare the expression level of tap and p55 eux pump genes in non mutated isolates, mutated isolates in rpoB genes and susceptible isolates.


Abstract Background
The increasing drug resistance in Mycobacterium tuberculosis isolates has become a global problem for tuberculosis therapy programs. Genetic mutations in rifampin (RIF), one of the key drugs in the treatment of tuberculosis are main mechanism of resistant to this drug in M. tuberculosis. Absence of mutation in target genes, other mechanisms such as e ux pump suggests possible role of drug resistant.
The objective of this study was to nd out mutations in rpoB genes in rifampin resistant isolates and to compare the expression level of tap and p55 e ux pump genes in non mutated isolates, mutated isolates in rpoB genes and susceptible isolates.

Methods
In this study, antimicrobial sensitivity test on rst line drugs was performed on 200 M. tuberculosis isolates, obtained from TB center in Shiraz (IRAN) and genetic mutations were evaluated in rpoB gene in RIF resistant isolates by multiplex PCR, followed expression level evaluated by Real-time PCR.
The expression level of tap and p55 genes was considerably higher in resistant isolates which had no mutation compared to the expression level of genes in the isolates which had mutation in target genes.

Conclusion
The accumulating data suggest the probable role of e ux pump in M. tuberculosis drug resistance, the validation of data needs further phenotypic assays of these pumps. Background Tuberculosis (TB) is a deadly infectious disease which remains one of the most important health problems in world wide [1].
According to the World Health Organization (WHO) reports in 2019, there were 7 million new cases of TB and 1.2 million death caused by TB in 2018 [2].
The increasing prevalence of drug resistant in Mycobacterium tuberculosis (MTB) strains is a major challenge to the control and treatment of TB. One of the forms of drug resistance tuberculosis is multidrug resistant tuberculosis (MDR-TB) (strains resistant to at least two rst-line drugs, namely, isoniazid (INH) and rifampin (RIF)), which emergence of MDR-TB are major problem to management of TB [3,4] and factors such as poor drug quality, inappropriate administration of anti-TB drugs and inadequate treatment regimens, inappropriate and incomplete drug use by the patient and previous treatment of TB are the most important factors in increasing the prevalence of drug resistant strain [5].
RIF is one of the key and important rst line antibiotics, which is used together with INH as the main anti-TB regimen. Since rifampin resistant M. tuberculosis isolates are often also resistant to INH and monoresistance to rifampin is rare, therefore, rapid identi cation of rifampin-resistant isolates is important because rifampin resistance is a marker for MDR-TB, which is a major barrier to the treatment of TB [6].
RIF binds to the β-subunit of the RNA polymerase and inhibits elongation of mRNA. The main mechanism of resistance to rifampin is through the mutation in the rpoB gene, which encodes the βsubunit of the RNA polymerase [7,8], and according to previous studies, the most frequently mutations in RIF-resistant clinical isolates, occur in the region of 81 base pairs of rpoB gene is called the Rifampin resistance determining region (RRDR), and mutations in codons 531, 526 and 516 are the most commonly reported mutations in this region However, roughly 5% of M. tuberculosis clinical isolates resistant to RIF have no mutation in the rpoB gene, and it increases the possibility of other mechanisms, such as activity e ux pumps, in the emergence of drug resistance [9,10].
The e ux pumps are membrane proteins that can remove various substrates, including drugs, from the cell [11,12]. The rst e ux pump known in Mycobacteria was LfrA in Mycobacterium. smegmatis, which confer resistance to different compounds including ethidium bromide, acridine, uoroquinolones, and some quaternary ammonium compounds [10]. The bacterial e ux pumps are divided into 5 families: 1the major facilitator superfamily, 2-the small multidrug resistance, 3-the resistance nodulation cell division, 4-the multidrug and toxic compound extrusion, which use proton motive force and 5-the ATP binding cassette superfamily, which uses ATP as an energy source to remove drugs from the cell [13,14].
Few studies to date have investigated the role and the extent of the expression of the e ux pump genes in the development of drug resistance in M. tuberculosis clinical isolates and most studies have investigated the e ux pumps genes by using of laboratory strains (Mycobacterium bovis, Mycobacterium smegmatis) and the transfer of hypothetical e ux pump genes to the strains listed as hosts and the use of inhibitors of e ux pumps [15]. Considering the high prevalence of M. tuberculosis and the increased drug resistant strains, as well as the importance of rifampin as the rst line antituberculosis drug, it is important to study the mechanism of resistance to the drug mentioned in the isolates of M. tuberculosis. The aim of this study was to investigate the prevalence of mutations in codons 516, 526 and 531 of the rpoB gene in M. tuberculosis isolates, and to determine the expression level of the two e ux pumps genes Rv1410c (p55) and Rv1258c (tap) and compare their expression level in RIF resistant M. tuberculosis clinical isolates had mutation in the rpoB gene, RIF resistant isolates had no mutation in the rpoB gene and also in the drug-susceptible isolate. Both selected gene were selected from the MFS family and based on previous studies [10,14].

Methods
Isolation and drug susceptibility testing: In this study, 200 M. tuberculosis isolates were obtained from TB center of shiraz and molecular assay were done to con rm MTB isolates and con rmed strains were included this study.
Antibiotic susceptibility test against rst-line drugs was performed on MTB isolates by the proportional method on the Lowenstein-Jensen medium according to the WHO recommendation. The concentrations of tested anti tuberculosis drugs were as follows: INH 0.2 mg/mL, RIF 1.0 mg/m, streptomycin (STR) 2.0 mg/mL and ethambutol (EMB) 5.0 mg/ml.

DNA extraction:
Genomic bacterial DNA was extracted from MTB isolates by DNA extraction kit (QIA Gen). To determine the DNA concentration in the extracted specimen, the optical absorption at 260 and 280 nm wavelengths were measured by spectrophotometer, if the absorbance ratio of 260 to 280 nanometers was more than 1.8, then it was considered appropriate for PCR.

Ampli cation PCR:
Ampli cation of 16s rRNA genes to con rm MTB isolates were done with speci c primers shown in Table  1. mutations in the rpoB gene (in common codons) were investigated in RIF resistant isolates by Multiplex PCR and using the speci c primers shown in Table 1.
Ampli cation of the rpoB gene was conducted as: an initial denaturation at 94º for 5 min followed by 30 cycles of denaturation at 94º for 30 sec , 40 sec of annealing and extension at 72°C for 50 sec and nal extension at 72°C for 5 min and reaction mixture were prepared in a total volume of 25µl contained 2.5 µl of 1X PCR buffer, 1 µl of 2 mM dNTP, 1 µl of each primer, 0.25 µl Taq DNA polymerase and 2 µl of extracted DNA.
RNA extraction, cDNA synthesis and reverse transcription: Total RNA extraction was carried out from Subcultured MTB isolates in LJ medium using QIA Gen RNA extraction kit according to the manufacturer s protocol. cDNA synthesis was performed using First Strand cDNA synthesis kit (BIONEER, Korea) according to the manufacturer s instructions. Then, Reverse Transcriptase-PCR was performed to assay the expression of the e ux pumps genes (tap, p55) using the primers listed in table 1. The thermal cycling conditions included: initial denaturation at 95°C for 10 min followed by 35 cycles at 95°C for 35 sec, 62°C for 30 sec and 72°C for 45 sec and the nal extension at 72° C for 10 min.
Quanti cation of gene expression using real-time quantitative PCR (qRT-PCR): Reaction mixtures were prepared using 12.5µl of 2X SYBR Green master mix, 1µl of each primer, 3µl cDNA and 7µl RNase-free water to a nal volume of 25µl and the thermal cycling conditions were set at: 1 cycles of initial denaturation at 95ºC for 2 min followed by 35 cycles of denaturation at 95ºC for 25 sec, annealing at 62ºC for 15 min and extension at 72ºC for 15 min and the last step at 4ºC for 2 min.In this method, the expression of 16s rRNA gene as a House Keeping Gene with a constant expression as control was used to compare the expression of the genes by Ratio Quanti cation Method and through normalization against unit mass. In this method, the initial values of the desired gene cDNA and the 16s rRNA gene were determined by measuring OD and from each gene were used to an equal ratio in real time PCR. After real time PCR, the Ct of control and sample gene was measured and their ratio was calculated. The standard curve line gradient for tap and p55 genes and reaction e ciency was calculated by the software using the formula E = 10 (-1slope) . The amount of ΔCt for each gene was obtained through the formula ΔCt = Ct (sample gene -control gene) and the gene ratio was calculated using the formula Ratio = E (ΔCt) .

Results
The results of drug susceptibility testing of M. tuberculosis isolates: The results of mutations in rpoB gene in rifampin-resistant isolates: In the present study, PCR results revealed that 12 (52.17%) isolates out of the 23 RIF resistant MTB isolates, have mutations in the rpoB gene and out of them 5 (41.67%) strains in codon 531, 4 (33.33%) in codon 526 and 3 (25%) in codon 516 were mutated and were considered as RIF resistant genotypes isolates, and 11 (47.83%) isolates had no mutations in these codons and were considered as resistant phenotypes and RIF sensitive genotypes isolates.
Results of investigating of tap and p55 genes expression in rifampin-resistant isolates by Reverse Transcriptase -PCR method: from all 23 RIF resistant MTB samples, 16 (69.56%) isolates expressed tap gene, 10 (62.5%) isolates were related to RIF resistant phenotypes and non-mutated isolates, and 6 (37.5%) of them were related to RIF resistant isolates which have mutation in the rpoB gene and 15/23 (65.22%) isolates expressed P55 gene, and 9 (60%) of them were related to non-mutated isolates, and 6 (40%) of them were isolates which had mutations in the rpoB gene.
Results of investigating of tap and p55 genes expression level by Real time PCR method: considering that the reaction e ciency in this experiment was close to 100%, therefore, for the genes ratio to be obtained, the reaction e ciency was considered 2 and the control Ct for the tap gene is 25.23 and for the p55 gene, 36.76 is calculated.The threshold and standard chart of tap gene are shown in Supplements 1 and 2. Based on the results listed in the table 2, the average gene ratio of tap in nonmutated isolates was 6.85, in RIF resistant isolates with mutation was 0.54 and in RIF sensitive isolates was 0.64 (P <0.05). The standard charts of p55 gene also are shown in Supplements 3 and 4 and according to the results presented in the table 3, the average gene ratio of p55 in non-mutated isolates were 8.88, in resistant isolates with mutation were 0.84 and in sensitive isolates were 0.85 (P<0.05).

Discussion
The results of our study indicate that among 200 MTB clinical isolates after drug susceptibility testing, 67 (33.5%) were resistant to at least one of the rst line drugs and 23 (34.33%) isolates were rifampin resistant, and 19 (28.36%) were identi ed as MDR.
The higher prevalence of resistance to at least one of the rst-line drugs and the higher prevalence of MDR in comparison with our study, were reported from India (85.9%, 47.54%), Cameroon (81.3%, 50%) and pakistan (71.92%, 42.10%) [16]. A study of Nepal 2017 reported a relatively higher prevalence of resistance to one or more drugs (38.2%) in comparison with our study, but the prevalence of MDR (11.7%) and the resistance to RIF (15.7%) were lower in their investigation than the results of our survey [17]. Also in another study in Turkey in 2010 comparing to our study, a lower rate of prevalence resistance (28.7%), MDR (4.5%) and resistance to RIF (6.3%) were reported [18].
In the present study, we found that out of 23 RIF resistant isolates in the antibiotic susceptibility test, 12 (52.17%) isolates had mutations and 11 (47.83%) had no mutations in the rpoB gene. In our study, the highest frequency of mutations was found in codon 531 (41.67%), followed by that codon 526 (33.33%) and codon 516 (25%).
These ndings were similar to reported results in Egypt in 2009, which the frequency rate of mutation in codons of 531, 526, and 516, were reported 45%, 30% and 20%, respectively. [19] Moreover, in another similar study in Vietnam the frequency of mutation were reported in codons 531(43%), 526 (31%) and 516 (15%), and it was very close to the results of our study [20].
The high frequency of mutation in codon 531 was found in previous studies in some other countries, including Indonesia (59.09%) [21], Taiwan (68. Also, in different studies, the importance of the e ux pumps and the role of them in the extrude of the drug have been pointed out. It has been shown that there is a direct relationship between overexpression of different genes of the e ux pumps and the resistance to various drugs in mycobacteria [27].
In the present study about the e ux pumps, the expression of the tap and p55 e ux pump genes in RIF resistant MTB isolates were investigated by Reverse Transcriptase -PCR and also the level of expression of these genes were evaluated using qRT-PCR method.
The results of our study about the expression of these genes shows that out of the 23 rifampin-resistant isolates, among 11 isolates which had no mutations (11 non mutated isolates) in the rpoB gene, 10 (90.91%) have been expressed tap gene, and out of 12 isolates that have mutations, 6 (50%) have been expressed tap gene. In samples with the P55 e ux pump gene, 81.8% of the non-mutated isolates and 50% of the mutated isolates in the rpoB gene, have been expressed the p55 gene and the results of qRT-PCR in our study indicate that the expression level of the tap and p55 genes in resistant phenotypic and non-mutated samples compared to resistant and mutated samples and also sensitive samples were more than 10 fold and there was a signi cant difference in the level of expression in RIF resistant samples. This may indicate the expression of e ux pump genes in RIF resistant phenotypic isolates but had no mutation in the target genes was higher than that in resistant and mutated isolates and may be effective in resistance to this drug.
Similar studies have been done on clinical MTB isolates using qRT-PCR method, including a study by Guilian Li et al. in chine that showed out of 9 MDR isolates, 8 of them overexpressed in at least one of the 20 e ux pump genes of their investigation, and the expression level of 11 e ux pump genes out of 20 of their studied genes, including P55, were signi cantly higher in 9 MDR isolates than that in 10 drug susceptible isolates (P <0.05) [26].
In another similar study by Yamchi et al. in 2015, the expression levels of several genes, including p55 and jefA, in drug-resistant and drug-sensitive MTB and H37Rv reference strains have been investigated by qRT-PCR method. They showed that out of 21 drug resistant isolates, 12 (57.14%) have expressions more than 4 fold, and out of 7 isolates without any mutation in the drug target genes, 5 (71.43%) had overexpression in the e ux pump. In their study, the level of expression of the e ux pump genes in drugresistant and drug sensitive samples were signi cantly higher than the standard strain (P<0.05) [28]. Moreover, another study by Mustafa kerem calgin et al. in 2013 had been done that they reported the expression level of e ux pump genes including tap and p55 were signi cantly higher in multidrugresistant and drug-sensitive MTB isolates compared to standard strains. But, there were no signi cant differences between multidrug-resistant and drug sensitive isolates [14].
A study in India by Anshika Narang et al. in 2019 have been investigated the expression of several e ux pump genes in RIF resistance and RIF susceptible MTB isolates. in their study no correlation was evident between examined e ux pumps and RIF resistance although their study limited to a small number of clinical isolates, overexpression of 2 genes from tested e ux pumps genes (mmpL5 and Rv0194) in a greater number of RIF resistant isolates as compared to RIF susceptible isolates suggests a role in RIF resistance [29].

Conclusions
In the present study, out of 23 RIF resistant isolates, 11 (47.83%) had no mutations in the rpoB gene, which could indicate the role of other mechanisms such as e ux Pump systems. According to the results of our study, the expression level of the studied e ux pump genes in resistant and non-mutated MTB isolates was signi cantly higher than mutated isolates and susceptible isolate. The results increase the likelihood of this hypothesis that in isolates with phenotypic resistance but not mutations in the drug target genes, it may be that the a ux pumps to be effective in resistance, although determining its roles requires further studies and the evaluation of the phenotypic activity of these pumps.  Rv1258c-R 5´AAT ACT TCA CCG ACC ACC AGC AAC 3′ Rv1410c-F 5´TCT TGG ACT TCC GGT TCA TC 3′ 221 [14] Rv1410c-R 5´TAC CGG TTC AAC CAG ATC CTG 3′