Frequency and Molecular Characterization of Rifampicin-Resistance in rpoB Region of Multiple Drug Resistance (MDR) Isolates from Tuberculosis Patients in Southern Endemic Region of Iran

INTRODUCTION

The World Health Organization (WHO) has estimated that one-third of the world’s population, approximately 2 billion people have been infected with MTB. Five years ago in Iran, prevalence rate of tuberculosis (TB) was reported to be as high as 17 in 100,000 (Zaker et al., 2006). Recent worldwide surveillance has demonstrated that drug-resistant strains are now widespread and reaching alarmingly high levels in certain countries. Multiple drug resistance (MDR) TB is a potentially untreatable and transmissible disease associated with high mortality. Zabol is an endemic region in the south of Iran (Afghanistan border) with 10 to 13%  MDR-TB among 141 TB cases per 100,000 populations (Zaker et al., 2006). Resistance to rifampicin is increasing because of its widespread application, resulting in the selection of mutants resistant to other components of short-course chemotherapy. For example, 88% of hospitalized patients with drug resistant TB admitted to Massih Daneshvari hospital (Tehran-Iran) proved to be resistant to at least isoniazid and rifampicin (Namaei et al., 2006).
In bacterial populations, the generation of antibiotic resistance depends on the rate of emergence of resistant mutants (Mokrousov et al., 2003). A correlation between high mutation rate, antibiotic resistance and virulence in bacteria has been reported in several studies (Valim et al., 2000). The detection of resistant MTB strains is generally performed by the conventional susceptibility method which requires culturing the bacilli in the presence of different drugs. The rapid detection of rifampicin-resistant strains is particularly important, since it also represents a valuable surrogate marker for MDR-TB resistance, which is a tremendous obstacle to TB therapy (Sajduda et al., 2004; Kapur et al., 1994). Collectively, DNA sequencing studies have demonstrated that more than 95% of rifampicin-resistant MTB strains have a mutation within the 81bp hot-spot region (codons 507 to 533) of the RNA polymerase b-subunit (rpoB) gene (Sajduda et al., 2004). The prevalence of the mutations determined so far varies for MTB strains obtained from different countries. Recently, the molecular basis of rifampicin resistance in MTB was identified (Telenti et al., 1997). Thus, it is important to determine the distribution of resistance mutations in each country prior to molecular tests being introduced for routine diagnostics (Sajduda et al., 2004; Kim et al., 2004; Mani et al., 2001). Drug resistance has been known since the discovery of the first anti-TB drug, streptomycin in the mid 1940s and the presence of resistant mutants in wild populations of mycobacteria have been well documented (Lin et al., 2004; Mikhailovich et al., 2001; Musser et al., 1995; Williams et al., 1994).
Genotypic assays which detect mutations within the rpoB regions are predictive of drug resistance and have the potential to provide a rapid method for detection of isoniazid-resistant isolates.The aim of this study was to determine resistance-associated mutations in the 81 bp region of the rpoB gene in 34 rifampicin-resistant MTB strains from Iranian TB patients.

MATERIALS AND METHODS

Mycobacterium tuberculosis (MTB) isolates: From December 2005 to May 2006, MTB isolates were obtained from sputum samples of patients with active pulmonary tuberculosis (TB) in Zabol, the southern endemic region of Iran. All 91 TB patients had proven registration of clinical diagnostic examinations, such as chest X-ray, purified protein derivative (PPD), cough, weight loss, gender, etc. The isolates were cultured on Lowenstein-Jensen solid medium and the resulting colonies were identified at the species level using 2-thiophene carboxylic acid (TCH) and paranitrobenzoic acid (PN99B) selective media, or by standard biochemical procedures. Two sensitive isolates were used as negative controls.
 
Susceptibility testing: Anti-microbial drug susceptibility testing (AMST) was performed using the CDC standard conventional proportional method. This involved the use of rifampicin (Rif) 40 mg/ml, isoniazid (INH) 0.2 mg/ml, streptomycin (SM) 4 mg/ml, and kanamycin (K) 20 mg/ml in  Lowen stein-Jensen medium. in addition, breakpoint concentrations of isoniazid 0.1 mg/ml, and rifampicin 2.0 mg/ml were also used in the BACTEC system. Mutations of the rpoB gene were identified in 34 rifampicin resistant isolates by DNA sequencing. AMST was performed following sequencing to confirm resistance using different concentrations of rifampicin (50, 75 and 100 mg/ml) in the Lowen stein-Jensen medium.

PCR amplification: DNA extraction was carried out using Fermentas kit’s (K512). A 411-bp fragment of the rpoB gene was amplified by PCR with primers rpoB-F (5´-TACGGTCGGCGAGCTGATCC-3´) and rpoB-R (5´-TACGGCGTTTCGATGAACC-3´) (Miriam et al., 2001). PCR reaction was performed in a 50 ml reaction mixture containing 50 mM KCl, 10 mM Tris (pH 8.0), 1.5 mM MgCl2, 5 mM of deoxynucleoside triphosphates (dNTPs), 1U of Taq polymerase, 20 pmoles of each set of primers and 6 mM of chromosomal DNA. Samples were then subjected to one cycle of denaturation at 94ºC for 5 min, followed by 36 cycles at 94ºC for 1 min, 57ºC for 1 min, 72ºC for 1 min and a final cycle at 72ºC for 10 min to complete the elongation of the PCR intermediate products. PCR products were then run on 2% agarose gels and examined for the presence of the 411-bp band after ethidium bromide staining. The DNA purification was performed using a Sigma kit          (USA).

DNA Sequencing: A 411bp fragment of the rpoB gene, containing the 81 bp rpoB fragment, was amplified by PCR using two primers: rpoB-F (5´-TACGGTCGGCGAGCTGATCC-3´) or rpoB-R (5´-TACGGCGTTTCGATGAACC-3´). PCR was carried out in a 8 ml reaction mixture containing 0.25  ml of DNA polymerase in 0.9 ml of buffer (PCR), 2 ml of a mixture of dNTP and dNNTP (dATP, dTTP, dCTP, dGTP), 0.5 ml of each primer (2.5 pmoles), 1 ml of DNA and 3.35 ml of H2O (Molecular Biology grade). Amplification was carried out for 33 cycles, with the following programme: denaturation at 94ºC for 30 sec; primer annealing at 54ºC for 30 sec; extension at 72ºC for 90 sec. A 411 bp fragment of the rpoB gene extracted from MTB strains was sequenced by the Amersham auto sequencer and Amersham Pharmacia DYEnamic ET Terminator Cycle Sequencing Premix Kits. Alignment of the DNA fragments (rpoB) was performed with the help of the MEGA 3.1 software.
Data analysis of DNA sequences: Alignment of the DNA fragments (rpoB) was carried out with the help of MEGA 3.1 and DNAMAN softwares and compared with standard strains CDC1551, H37RV and MTB strain 210. The BLAST 2 sequences program was used for DNA sequence comparisons (http://www.ncbi.nlm.nih.gov /BLAST/).
RESULTS

Bacterial strains and drug susceptibility assay: All samples were cultured and identified as MTB by the PCR method. All 34 isolates examined were resistant to rifampicin. But 11 (34%), 28 (90%) and 10 (31%) of the isolates were found to be resistant to isoniazid, streptomycin and etambutol, respectively. In this study we found four strains to be mono-resistant to rifampicin. From 34 rifampicin resistant isolates, 12 (35%) were isolated from sputum of patients with primary infection and 22 (65%) isolates were obtained from secondary infections.
   Definitions in this study: primary infection is referred to a patient who does not have a previous history of TB disease nor medical treatment. Secondary infection demonstrates a previos history of TB disease in the patient’s medical records.

PCR amplification and DNA sequencing: In 29 rifampicin-resistant MTB strains (85%), 60 mutations and 13 micro-deletions were identified. In 5 (15%) rifampicin resistant MTB isolates, no mutations were found in the core region of the rpoB gene. Of 60  mutations identified, 6 were silent (8.3%) and 54 (91.7%) were missense. Most of detected deletions were located in codons 510 GAG/_AG (12.5%). All silent mutations were localized in codon 507, while missense mutations revealed 23 types of amino acid substitutions. Most frequent mutated codons in the Iranian strains were codon 523 (GGG"GG_, GGG"GCG) and codon 526 (CAC"TAC, CAC"CGC, CAC"AAC, CAC"TTC, CAC"CAA, CAC"_GC) indicating six types of mutations (Tables 1 and 2). Mutations in codons 510, 507 and 531 were observed in 27%, 24% and 21% of isolates, respectively. Correspondingly, mutations in codon 523 resulted in Gly523Ala replacement and in codon 531, Ser531Leu and Ser531Phe. Six alleles were observed in codon 526, and 3 alleles in triplets 507, 508 and 513. In 6 strains (18%) single mutations were located in codons 526 and 510, while isolates with multiple mutations revealed double (34%), triple (22%) and quadruple (3%) mutations. 12% of the strains harboured 5 mutations (Tables 1 and 2).
We detected deletion mutations in codons 510, 520, 523, 526 and 527, stop mutation in codon 513 and silent mutation in codon 507 (Table 1).

DISCUSSION

The rpoB codons 531, 526, 516 and 511 are the most frequently mutated sites, observed worldwide. However, variations in the relative frequencies of mutations in these codons have been described for isolates from different geographic locations (Bakonyte et al., 2005; Marin et al., 2004; Matsiota et al., 1998). Other studies have also indicated that these mutations are the most prevalent worldwide (Namaei et al., 2006; Kapur et al., 1994; Bakonyte et al., 2005). These differences reflect the complex and crucial interactions between the drug and its target at the molecular level where the position of the affected allele seems to be variable. This finding is not in agreement with other authors who have reported different levels of high (Pozzi et al., 1999; Hirano et al., 1999; Sifuentes et al., 1995) and low (Namaei et al., 2006; Hirano et al., 1999) resistance associated with specific nucleotide replacements. All rifampicin resistant isolates studied had mutations of different types in the rpoB region, however the relationship between the combination of specific types of mutations and rifampicin resistance is unclear. This is the first report describing the genetic characteristics of multidrug-resistant MTB strains isolated from TB-patients in Iran. The finding of mutations is partially comparable and resembles those strains reported in other countries (India, Russia, China, USA and Lithuania). CAG mutation of codon 510 (deletion or CTG or CAC or CAT) is very seldom detected in other countries. However in this study (Table 1) a higher number of deletion mutations (9 strains) with respect to one base C ( _AG) were found. On the other hand, in other countries no changes in codon 510 (1, 5, 15) have been observed. Mutation CAG"CAT has been found in India (Mani et al., 2001), –CAG"CAT in Russia, and CAG"GAG, TAG  in Belarus, CAG"GAG in Lithuania and CAG"GAG in Poland, for the same codon (Namaei et al., 2006; Bakonyte et al., 2005; Mokrousov et al., 2003; Telenti et al., 1997). The important findings of this study revealed that codons 510 (12.51%), 523 (23.6%) and 526 (16.6%) had the most frequent occurrence of mutation bearing sites. Infact mutations in codons 531 and 526 occur most frequently in the world (TCG"TTG for codon 531, and CAC"TAC for 526) (Namaei et al., 2006; Bakonyte et al., 2005; Lishi et al., 2002; Sifuentes-Osornio et al., 1995). The data of this study are very closely related to those observed in Asia (60%). Comparison of our data with other countries indicate fewer mutations in codon 531 (TCG"TTG) (Ruiz et al., 2004; Lilly et al., 1999; Yuen et al., 1999) and more mutations in codon 526 (CAC"TAC, CAC"_GC, CAC"CGC, CAC"AAC, CAC"TTC and CAC"CAA) (Valim et al., 2000; Huang et al., 2002; Matsiota-Bernard et al., 1998; Williams et al., 1998). Mutations in codon 526 (CAC"CAG) and codon 516 (GAC"GTC) not often seen in Iran are usually observed in Poland and USA (Sajduda et al., 2004; Kim et al., 2004; McCammon et al., 2005., Yun et al., 2005). In this investigation mutations in codon 511, representing one of the frequent mutations worldwide (Tables 1 and 2) were also observed. The high percentage of double mutations found among Iranian strains (32%) differed clearly with the lower prevalence of double mutations in other studies (Namaei et al., 2006; Williams et al., 1994; Lishi et al., 2002; Barfai et al., 2001). Noticeable findings of this study indicate the high frequency of double (32%), triple (20%) and quadruple (2.9%) mutations occurring in separate codons. It should be noted that five phenotypic rifampicin resistance strains revealed no mutations. The combination of two single point mutations has been described previously for rifampicin-resistant isolates of MTB (Pozzi et al., 1999; Kapur et al., 1994). Silent mutations 6 (17.6%) were also detected in 6 different isolates which demonstrated an absence of drug resistance to rifampicin. These 6 isolates consisted of quadruple mutations amongst which one silent mutation was detected in codon 507. Twenty-two isolates (65%) were collected from secondary cases (data not shown).
  Of 34 rifampicin resistant isolates, 12 (35%) obtained from the sputum of patients with primary infection, consisted of 14 different types of mutations.  predominant mutations were demonstrated by 4 isolates (28.5%) in codon 526, 3 (21.4%) in codon 523, 2 (14.2%) in codon 510, and the remaining codons showed no significant frequency of mutations (Tables 1 and 2). The 22 (65%) isolates obtained from secondary infection contained 59 different types of mutations. From these 8 isolates (13.5%) showed mutations in codon 526, 14 (23.7%) in codon 523 and 7 (11.8%) in codon 510.
This study indicates multiple mutations in codon 523 and codon 526 among the MDR strains of MTB collected from sputum of patients bearing secondary infections. It also demonstrates that the highest frequency of mutations in codons 523 and 526, is observed in both primary and secondary infections, in  the southern endemic border of Iran. Although different mutations have been reported for the rpoB gene of MTB by PCR-SSCP, but it may not be a reliable tool for the detection of resistance to rifampicin in this strain (Miriam et al., 2001). However, if a strong correlation between specific mutations and the level of resistance is confirmed in other settings, the level of rifampicin-resistance may be predictable by DNA sequence-based resistance detection methods (Miriam et al., 2001). In this study the detection of deletion mutations (in codons 510, 520, 523, 526 and 527), stop mutation (in codon 513) and silent mutation (in codon 507) (Table 1) confirmed results by Van Der Zanden  et al. (2003).

Acknowledgments

We thank our colleagues from the Institute of Tuberculosis and pulmonary of Belarus (Dr. Evgeni Romanovich) for providing clinical strains and valuable advice.