strains of Mycobacterium tuberculosis isolates from Belarusian patients
Saeed ZAKER BOSTANABAD2,3, Leonid P. TITOV1,2, Veranika V. SLIZEN1,2, Mohammad TAGHIKHANI3,4, Ahmadreza BAHRMAND3
1 Belarusian Research Institute for Epidemiology and Microbiology, Minsk, Belarus,
2Belarusian State Medical University, Belarus,
3Pasteur Institute of Iran, Iran,
4Biochemistry Department, School of Medical Sciences Tarbiat Modarres University.
ÖZET
Belaruslu hastalardan izole edilen izoniazide dirençli Mycobacterium tuberculosis’lerdeki katG mutasyonları
Bu çalışmada Belaruslu hastalardan izole edilen Mycobacterium tuberculosis’te katG mutasyonlarının sıklığı, tipi ve loka- lizasyonunu belirlemeyi hedefledik. Belaruslu hastalarda katalaz peroksidaz (katG) genindeki iki farklı kodona ait mutas- yonların izoniazide karşı direnç gelişimine yol açtığı gösterildi. Belarus’un farklı bölgelerinden tolam 42 Rif-r ve Inhr (MDR) zinciri elde edildi. Yapılan kültür duyarlılık testlerinde bütün bu 42 zincirin de streptomisine (%90) dirençli olduğu, 16 zin- cirin ise etambutole (%43) dirençli olduğu bulundu. DNA ekstraksiyonu, standart PCR ve katG geni amplifikasyonu yön- temleri kullanıldı. En fazla etkilenen katG kodonları 315 (%95), 316 (%16.2) ve 309 (%14.5) idi. 315 numaralı kodonda dört tip mutasyon görüldü: AGC→ACC (n= 36) %32.4, AGC→AGG (n= 1) %0.9, AGC→AAC (n= 2) %1.8, AGC→GGC (n= 1) %0.9.
316 numaralı kodonda bir tane mutasyon görülürken; GGC→AGC (n= 18) %16.2, 309 no’lu kodonda toplam 4 mutasyon vardı: GGT→GGT (n= 7) %6.3, GGT→GCT (n= 4) %3.6, GGT→GTC (n= 3) %2.7, GGT→GGG (n= 1) %0.9. Gösterilen mutas- yonların %34’ü 309 no’lu kodona, %37’si 316 no’lu kodona, %29’u ise diğer kodonlara aitti. 463 ve 35 numaralı kodonlar- da 2 farklı zincir mutasyonu bulunurken, 454 ve 357 numaralı kodonlarda ise hiçbir mutasyona rastlanmamıştır. Sonuç olarak bütün INH-r MBT’lerdeki direnç 315 no’lu kodondaki nükleotid değişiklikleri ile ilişkilidir.
Anahtar Kelimeler: Belarus’ta katG geni mutasyon sıklığı.
Yazışma Adresi (Address for Correspondence):
Saeed ZAKER BOSTANABAD, MD, Belarusian Research Institute for Epidemiology and Microbiology, Minsk, BELARUS
e-mail: saeedzaker20@yahoo.com
Resistance to isoniazid is associated with a vari- ety of mutations affecting one or more genes such as those encoding catalase-peroxidase (katG) (1-5). Isoniazid (INH) is the first line che- motherapeutic drugs used in tuberculosis (TB) therapy (6-9). KatG gene is the most commonly targeted region with majority of mutations oc- curring in codon 315 in 30-90% of INH-resistant strains, depending on geographical distribution (1,3,10-12). The observation that most INH-re- sistant Mycobacterium tuberculosis strains did not have gross katG deletions suggested the ne- ed to more precisely analyze the structure of katG present in resistant organisms. Several groups have now reported that many IHN-resis- tant strains contain missense and other types of mutations (12-17). KatG gene is the most com- monly targeted region with majority of mutati- ons occurring in codon 315 in 30-90% of INH- resistant strains depending on geographical dist-
ribution and extensive studies have demonstra- ted that INH resistance is most frequency asso- ciated with a specific mutation in katG (codon 315), a gene that encodes the katG enzyme in M. tuberculosis (3,5,18,19). Mutations in several genes and genomic regions of M. tuberculosis are involved in the occurrence of resistance to INH (19-23). Another frequent target is the re- gulatory region of the inhA gene, where mutati- ons have been reported in up to 32% of INH-re- sistant isolates (10,12,14,16). Mutations in ot- her genomic regions, such as the promoter of ahpC gene and in the kasA gene, have been re- ported in 12-24% and 10-14% of INH-resistant strains, respectively (14,18,19). Genotypic as- says that detect mutations within such regions are predictive of drug resistance and have the potential to provide rapid detection of resistance in mycobacterial isolates. During the past few years several genotypic assays have been deve- SUMMARY
katG mutations in isoniazid-resistant strains of Mycobacterium tuberculosis isolates from Belarusian patients
Saeed ZAKER BOSTANABAD2,3, Leonid P. TITOV1,2, Veranika V. SLIZEN1,2, Mohammad TAGHIKHANI3,4, Ahmadreza BAHRMAND3
1 Belarusian Research Institute for Epidemiology and Microbiology, Minsk, Belarus,
2Belarusian State Medical University, Belarus,
3Pasteur Institute of Iran, Iran,
4Biochemistry Department, School of Medical Sciences Tarbiat Modarres University.
The aim of this study was to investigate the frequency, location and type of katG mutations in Mycobacterium tuberculosis strains isolated from patients in Belarus. Mutations in different codons causing resistance to isoniazide in the gene of catalase peroxides (katG) in Belarusian strains was determined. 42 strains of Rif-r and Inhr(MDR) were isolated in different regions of Belarus. Culture susceptibility testing of all 42 strains revealed resistance to streptomycin (90%), 16 stra- ins (43%) were resistant to etambutol. DNA Extraction, Standard PCR identification and katG gene amplification were per- formed. The most affected codons of katG gene were 315(95%), 316(16.2%), and 309(14.5%). Four types of mutations we- re identified in codon 315: AGC→ACC (n=36)32.4%, AGC→AGG (n=1) 0.9%, AGC→AAC (n=2) 1.8%, AGC→GGC (n=1) 0.9%. One type of mutation was found in codon 316: GGC→AGC (n=18)16.2%, four types of mutations were detected in co- don 309: GGT→GGT (n=7)6.3%, GGT→GCT (n=4)3.6%, GGT→GTC (n=3)2.7%, GGT→GGG (n=1)0.9%. Mutations in codon 309 make up 34%, in codon 316 (37%) and other types of mutations 29% of all detected mutations. In 2 isolated strains mu- tation were identified in codons 463, 35 and, in codons 454, 357 respectively and 2 isolates, there wasn’t fount any muta- tions. Concluding, all INH–r MBT had resistance-associated nucleotide changes mostly in codons 315.
Key Words: Frequency of mutation katG gene in Belarus.
loped for detection of the mutations responsible for INH resistance, particularly those in codon 315 of the katG gene (1,7,10,18). Resistance to isoniazide has the second degree of significance in Belarus (21,22). Scope of this work was to determine mutations in codons causing resis- tance to isoniazide in the gene of katG in Bela- rusian strains.
MATERIALS and METHODS Mycobacterial Strains
42 isoniazide-resistant strains isolated from spu- tum of patients with active pulmonary tubercu- losis were obtained from various geographic re- gions of Belarus from December 2004 to May 2005. All the strains were cultured on Lowenste- in-Jensen solid medium and grown colonies we- re identified to the species level using TCH (2- thiophene carboxylic acid) and PN99B (paranit- robenzoic acid) selective media or by standard biochemical procedures (for control growth M.
tuberculosis that non-growth in these culture).
Susceptibility, Methods and MICs
Drug susceptibility testing were performed using proportional method (isoniazid 1 µg/mL, rifam- pin 40 µg/mL, streptomycin 10 µg/mL, or et- hambutol 2 µg/mL) with absolute concentration of antituberculosis agent on slants using H37RV strain as positive control and additionally using the BACTEC system. In the absolute concentra- tion method, resistance was defined as growth on solid media containing graded concentrations of drugs with more than 20 CFU at a specific drug concentration. The breakpoints for INH were 1 µg/mL on Lowenstein-Jensen medium and 0.1 µg/mL on the BACTEC system and for RIF were 40.0 µg/mL on Lowenstein-Jensen medium and 2.0 µg/mL on the BACTEC system.
Standard PCR Identification and katG Gene Amplification
42 isoniazid-resistant isolates were collected and DNA extraction was performed according to (TAE 200 + 5% chelex100 {45 min 95°C}). DNA isolated from standards isolates of M. tuberculo- sis Academia and Mycobacterium bovis BCG were used as controls and all of isolates were checked with DNA kit technology for M. tuber-
culosis. A 209 bp segment of the katG gene was amplified by PCR using the following synthetic oligonucleotide primers: katG F 5-GAAA- CAGCGGCGCTGGATCGT-3, katG R 5- GTTGTCCCATTTCGTCGGGG-3. The following thermocycler parameters were applied with ini- tial denaturation at 95°C for 5 min; 36 cycles of denaturation at 94°C for 1 min; primer annealing at 56°C for 1 min; extension at 72°C for 1 min;
and a final extension at 72°C for 10 min. The PCR product was amplified and purified again and controlled on the gel for electrophoresis.
The final purified DNA was used for sequencing.
PCR-RFLP
Amplification was performed on segment 750 bp of katG region for all isolates by using pri- mers katG F 5-CGGGATCCGCTGGAGCA- GATGGGC-3 and katG R 5-CGGAATTC- CAGGGTGCGAATGACCT-3. Enzyme hpa II was used for restriction on segment 750 bp at 37°C for 120 min and 65°C for 20 min.
DNA Sequencing
The fragment 209 bp of the katG gene was se- quenced after amplification by PCR to analyze the mutations associated with INH resistance.
Template DNA for PCR was obtained using cetyltrimethylammonium bromide. A 209-bp fragment of katG gene was amplified by using primers katG F 5-GAAACAGCGGCGCTG- GATCGT-3 to generate a 209-bp product, which was purified (fermentas kit 513) and used for sequencing. PCR was carried out in 50 µL con- taining 2 µL KCl, 2 µL Tris (pH 8.0), 1.5 µL MgCl2, 5 µL dNTP, 1UTaq polymerase, 27 µL water (for molecular method), 20 pmol of each primer and 6-10 µL of DNA template. In brief, 2 µL of exonuclease I (10 U/µL) and 2 µL of shrimp alkaline phosphatase (2 U/µL) were ad- ded to 15 µL of PCR amplification mixture, and the mixture was incubated at 37°C for 45 min.
The reaction mixture was inactivated by heating to 65°C for 20 min. Sequencing of the PCR pro- duct was done at 33 cycles of denaturation at 94°C for 30 sec; primer annealing at 48°C for 45 sec; extension at 60°C for 4 min with Amersham kit. The fragment of katG gene isolated from tu- berculosis strains was sequenced with H37RV control by using the Amersham auto sequencer
and Amersham Pharmacia DYEnamic ET Ter- minator Cycle Sequencing Premix Kits.
DNA Sequencing Analyzing
Alignment of the DNA fragments (katG) was carried out with the help of MEGA and DNAMAN software (Gen bank_ PUBMED/BLAST) and was compared with standard strain of CDC 1551, H37RV and M. tuberculosis 210. The Blast 2 se- quences sotfware program was used for DNA sequence comparisons (http://www.ncbi.nlm.
nih.gov/BLAST/).
RESULTS
Mycobacterial Strains and Susceptibility All 42 strains examined were resistant to isoni- azid, rifampicine, streptomycin (90%) and 16 strains (43%) were resistant to etambutol.
PCR and DNA Sequencing Analysis
Mutations were observed in affected codons of PCR product segment 209 bp of katG gene, 305, 306, 307, 314, 316, 321, 328, 315, 316, 357, 454, 463 and 309. Four types of mutations we- re identified in codon 315: AGC→ ACC (n= 36) 32.4%, AGC→ AGG (n= 1) 0.9%, AGC→ AAC (n= 2) 1.8%, AGC→ GGC (n= 1) 0.9%. One type of mutation was found in codon 316: GGC → AGC (n= 18) 16.2%, four types of mutations we- re detected in codon 309: GGT→ GGT (n= 7) 6.3%, GGT→ GCT (n= 4) 3.6%, GGT→ GTC (n=
3) 2.7%, GGT → GGG (n= 1) 0.9% (Table 1).
Mutations in codon 309 revealed 34%, in codon 316 - 37%, other types of mutations 29% of all detected mutations. Among all strains (n= 40) 95% of mutation were detected in codon 315.
Two type of mutations were seen in codon 357:
GAC→ CAC and GAC→ AAC. In addition two mutations were also observed in codon 463:
CGG→ CTG and 454 GAG → CGA. In all 42 strains, nine one mutation (19%), 16 - two mu- tations (40.47%), 9 - three mutations (21.5%), four - 4 mutations (9.5%) and 4 - five mutations (9.5%) were determined (Table 2). Two isolates did not have any mutations in katG gene.
PCR-RFLP
PCR-products 750 bp segment were cut with hpa II restriction enzyme and showed mutation in codon 309 katG gene (Figure 1).
Silent Mutations
Two silent mutations were detected in codons 306 (CCG → CCC) and 309 (GGT → GGG).
These silent mutations revealed no effect on the susceptibility pattern tested strains (Table 1).
DISCUSSION
The known genes related to INH-r are katG, in- hA, ahpC, kasA (5,10,14). Different articles re- ported that resistance of M. tuberculosis to iso- niazide is more corresponds to changes in co- don 315 (3,5,6,13,19,22). The most common mutations reported by other authors are in co- don 315 [AGC → ACC (Thr → Ser)] and 463 [CGG→ CTG (Arg→ Leu)] (3,5,6,13,19,23). We observed different number and types of mutati- ons: four in codons 315: AGC → ACC (Ser→ Thr) 32.4%, AGC → AGG (Ser → Arg) 0.9%, AGC→ AAC (Ser → Asn) 1.8%, AGC → GGC (Ser→ Gly) 0.9%, one in 463 CGG→ CTG (Arg
→ Leu) 0.9%, four in 309: GGT→ GTT (Cys→ Phe) 6.3%, GGT→ GCT (Cys→ Ser) 3.6%, GGT
→ GTC (Cys→ Phe) 2.7%, GGT→ GGG (Cys→ Thr) 0.9% and one in 316 GGC→ AGC (Gly→ Ser) 14.4%. Our data indicate that the highest number of mutations observed are 1: 315 AGC
→ ACC (Ser→ Thr) 32.4%, 2: 316 GGC→ AGC (Gly→ Ser) 14.4%. Similar results were reported in Latvia in where mutation in codon 315 of katG 95% corresponding to AGC→ ACC (Ser→ Thr) (90%) that resemble to our finding but AGC→ ACA (Ser→ Thr) (10%) that we have not obser- ved in our study (2). In Poland 90% of mutations were in codon 315 AGC which corresponds to 5 types of mutations (ACC, ACT, ACA, AAC, ATC) resembling similar pattern changes with Figure 1. 209-bp fragment of the katG segment amplified for purification and sequencing.
our data on nucleotide ACC and AAC. However in this research we did not observe nucleotide changes to ACT, ACA and ATC among Belaru- sian strains studied (21). In Russia highest 70%
of reported nucleotide changes were in codons 315 AGC→ ACC and in 463 CGC→ CTG which also shows similarity with our result (8,19,20).
Other mutations found in Belarusian strains can be important and effective cause of resistance because 60% of strains showed mutation in co- dons 316 and 309. Mutations detected in one strain in codons 463 and 357, 454 and 357 may indicate that this type of mutation in Belarus has less degree of concern when compared with ne-
ighbouring countries (1,19,20,23). In other stu- dies it was mentioned that silent mutations were never detected in katG gene (10,11,14,26). Ho- wever, in this research two silent mutations we- re obtained which indicate no effect on drug re- sistant pattern. We sequenced katG and rpoB genes of standard strain H37RV used as control, But non sense mutations were not observed in control strain. This work lets make a conclusion that sequencing is more sensitive for determina- tion of mutations among M.D.R. tuberculosis strains when compared with other methods such as spilotyping, SSCP and PCR-RFLP (16,17,23).
Table 1. Frequency of aminoacid and nucleotide changes of different codons in rrppooBBgene of 44 rifampicin-re- sistant strains of MM.. ttuubbeerrccuulloossiissisolated in Belarus.
Affected codon (number) Nucleotitede changes (number) Aminoacid changes
315 (n= 40) AGC→ ACC (n= 36) 32.4% Ser→ Thr
AGC→ AGG (n= 1) 0.9% Ser→ Arg AGC→ AAC (n= 2) 1.8% Ser→ Asn AGC→ GGC (n= 1) 0.9% Ser→ Gly
305 (n= 3) GGC→ GCC (n= 3) 2.7% Gly→ Ala
306 (n= 1) CCG→ CCC (n= 1) 0.9% Pro→ Pro
307 (n= 3) GGA→ CGA (n= 1) 0.9% Gly→ Arg
GGA→ GCA (n= 2) 1.8% Gly→ Ala
309 (n= 15) GGT→ GTT (n= 7) 6.3% Gly→ Cys
GGT→ GCT (n= 4) 3.6% Gly→ Ser GGT→ GTC (n= 3) 2.7% Gly→ Phe GGT→ GGG (n= 1) 0.9% Gly→ Gly
311 (n= 7) GAC→ TTC (n= 3) 2.7% Asp→ Phe
GAC→ TAC (n= 4) 3.6% Asp→ Tyr
314 (n= 3) ACC→ AAC (n= 2) 1.8% Ser→ Asn
ACC→ ACG (n= 1) 0.9% Ser→ Thr
316 (n= 18) GGC→ AGC (n= 18) 16.2% Gly→ Ser
321 (n= 6) TGG→ TAG (n= 2) 1.8% Trp→ Stop
TGG→ TTG (n= 2) 1.8% Trp→ Leu TGG→ TGT (n= 1) 0.9% Trp→ Cys TGG→ TCC (n= 1) 0.9% Trp→ Ser
328 (n= 4) TGG→ TGT (n= 4) 3.6% Trp→ Cys
463 (n= 1) CGG→ CTG (n= 1) 0.9% Arg→ Leu
357 (n= 2) GAC→ CAC (n= 1) 0.9% Asp→ His
GAC→ AAC (n= 1) 0.9% Asp→ Asn
454 (n= 2) GAG→ CGA (n= 2) 0.9% Glu→ Arg
The high percentage of double mutations found among the isolates in Belarus differed clearly from the lower prevalence of double mutations in other studies. A characteristic prominent fin- ding of this study was the high frequency of do- uble (40.47%) and triple (21.42%), quatriple (9.5%) and five mutations (9.5%) occurring in separate codons (Table 2). Two (1.9%) nonsen- se mutations were detected in 2 different (co- dons 306 and 309) isolates which demonstrated no effect of drug resistant pattern to isoniazid (Table 1). All isoniazid resistant isolates studied had mutations of differents types in katG region, however we don’t have any data of combination of specific types of mutations associated with isoniazid resistance. 32 (76%) were isolated from secondary cases (we don’t have data on reactivation cases. Interestingly all single and double mutations which were found in isolates of
sputum samples were taken from patients with secondary infection. From 42 isoniazid resistan- ce isolates 10 (24%) were from sputum of pati- ents with primary infection which consist of dif- ferent types of mutations. 4 isolates were found to have five mutation, in 4 isolates quartile and in 2 isolates triple which all sputum of patients with primary infection were detected mutation in codon 315. In 10 (23.8%) of patients with pri- mary infection shows to have predominant mu- tations in codon 315, in 5 (11.9%) in codon 316 and in 9 in codon 309, for patients with secon- dary infection 31 (49.2%) were found to have mutations in codon 315, 12 (19%) in codon 523 and 7 (11.1%) in codon 309 (Table 2). Isolates of M. tuberculosis isolated on the territory of Be- larus were characterized by the wide spectrum of the important mutations and might belong to the epidemic widespread clones.
Table 2. Kind and number of mutations in codones kkaattGGin MM.. ttuubbeerrccuulloossiissstrains isolated in Belarus.
Mutation Number of isolates Number of codon Frequency isolates Number of isolate
1 Mutation 8 (19%) 315 8 (19%) 94-457-932-489-
446-471-85-894
2 Mutations 17 (40.47%) 315-305 1 (2.38%) 411
315-309 3 (7.14%) 455-2331-469
315-311 2 (4.75%) 414-384
315-314 1 (2.38%) 118
315-316 8 (19.4%) Tub2-3255-24276-443-571 (1420)-3246-2262-23623
357-463 1 (2.38%) 402
357-454 1 (2.38%) 74
3 Mutations 9 (21.47%) 315-309-311 1 (2.38%) 139
315-307-309 1 (2.38%) 2331-2
315-305-321 1 (2.38%) 7285
315-305-309 1 (2.38%) 1416
315-316-328 1 (2.38%) 2738
315-316-321 2 (4.75%) 407-412
315-309-316 2 (4.75%) 139(2)-447
4 Mutations 4 (9.5%) 315-309-311-316 2 (4.75%) 369-370
315-309-316-328 1 (2.38%) 1217
315-309-321-328 1 (2.38%) 453
5 Mutations 4 (9.5%) 315-309-311-316-321 1 (2.38%) 2715
315-307-309-311-314 2 (4.75%) 388-368
315-316-306-321-328 1 (2.38%) 2831
Concluding that in Belarus the highest frequ- ency of common mutation shearing in between primary and secondary infections found in this study is occurring in codon 315.
ACKNOWLEDGEMENT
We thank our colleagues in Belarusian Institutes of Pulmonology and Tuberculosis for samples collections. This study was partially from Pasteur Institute of Iran and Belarusian Research Institute for Epidemiology and Microbiology, Minsk. We al- so thank Dr. M. Noghanian for analysis of data.
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