MICROBIAL DRUG RESISTANCE Volume 9, Number 1, 2003 © Mary Ann Liebert, Inc.
Frequency of Mutations in rpoB and Codons 315 and 463 of katG in Rifampin- and/or Isoniazid-Resistant Mycobacterium tuberculosis Isolates from Northeast Mexico JOSÉ MARÍA VIADER-SALVADÓ,1,4 CLAUDIA MARIBEL LUNA-AGUIRRE, 2 JORGE MAURICIO REYES-RUIZ,2 RAMÓN VALDEZ-LEAL, 3 MARIÁ DE LOS ANGELES DEL BOSQUE-MONCAYO, 3 ROLANDO TIJERINA-MENCHACA, 1 and MARTHA GUERRERO-OLAZARÁN1,4
ABSTRACT A total of 48 isoniazid (INH)- and rifampin (RIF)-resistant Mycobacterium tuberculosis isolates, 19 INH-resistant isolates, and 9 RIF-resistant isolates were randomly selected and tested for detecting mutations at codons 315 and 463 of katG by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and/or for detecting mutations at a 69-bp region of the rpoB gene by the INNO-LiPA Rif TB assay. Of the 67 INH-resistant isolates tested, 36 (53.7%) showed the mutation at codon 315 of katG; however, none of them showed the mutation at codon 463. The majority of the RIF-resistant samples analyzed (49 of 57, 86.0%) reacted positive with one of the four R-type probes. The R5-pattern (S531L mutation) was the most frequently observed (31 of 57, 54.4%), followed by R4a-pattern (H526Y mutation) 13 isolates (22.8%), R4b-pattern (H526D mutation) 4 isolates (7.0%), and R2-pattern (D516V mutation) 1 isolate (1.8%). Overall, there was agreement between the line probe kit and phenotypic RIF-susceptibility test for 56 (98.2%) of 57 RIF-resistant isolates tested. These results show that the mutation analysis at codon 315 of katG could be used as a screening assay prior to standard susceptibility testing, whereas mutations in the rpoB gene could be used successfully as genetic markers to rapidly detect RIF-resistant M. tuberculosis clinical isolates from northeast Mexico.
INTRODUCTION
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is conferred by specific point mutations and small insertions or deletions in a 69-bp region of the rpoB gene encoding for the b-subunit of mycobacterial RNA polymerase.25 Mexican laboratories that carry out susceptibility tests for M. tuberculosis mainly use the indirect proportion method, which requires a minimum of 3 weeks before susceptibility results are obtained from clinical isolates. However, due to the high case fatality rates of the present TB outbreak, the need for rapid identification and drug susceptibility testing of clinical isolates has increased. Genetic analysis of specific mutations involved in the resistance phenotype has been used as rapid tests for drug susceptibility.26 To detect mutations at codons 315 and 463 of the katG gene, the simple and rapid polymerase chain reactionrestriction fragment length polymorphism technique (PCRRFLP) using the restriction enzymes MspA1I and NciI has been
(TB) is a disease that has again become of great importance to public health due to the world-wide increase in the number of cases attributed in part to human immunodeficiency virus (HIV) coinfection and the emergence of drug-resistant Mycobacterium tuberculosis strains mainly to isoniazid (INH) and/or rifampin (RIF). Recently,24 a mutation frequency of 47–50% in the katG gene has been associated with M. tuberculosis resistance to INH, being the most frequently observed mutations at codons 315 and 463.17 However, a newer study has shown that the mutation at codon 463 is not related to resistance to INH.22 RIF is the second most frequent anti-TB drug to which M. tuberculosis shows resistance. Resistance to RIF in M. tuberculosis UBERCULOSIS
1 Departamento de Microbiología, 2 Departamento de Bioquímica, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, Nuevo León, México. 3 Laboratorio Estatal de Salud Pública de los Servicios de Salud de Nuevo León, Guadalupe, Nuevo León, México. 4 Departamento de Microbiología e Inmunología, Facultad de Ciencias Biológicas, Unidad C, U.A.N.L., 66450 San Nicolás de los Garza, N.L., México.
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used.8,11,14,23 On the other hand, the commercial line probe assay kit (INNO-LiPA Rif TB; Innogenetics, Zwijndrecht, Belgium), based on a reverse hybridization principle, detects point mutations in the 69-bp region of the rpoB gene.20 This line probe kit consists of five partially overlapping wild-type probes (S1–S5) that encompass the entire 69-bp region, four probes (R2, R4a, R4b, and R5) that specifically hybridize with amplicons carrying the four most frequently observed mutations (D516V, H526Y, H526D, and S531L, respectively), and one specific probe for the M. tuberculosis complex. If the mutations in the katG and/or rpoB genes involved with resistance to INH and/or RIF in northeast Mexico were the same as those that are reported for other countries, it would be possible to use these mutations as genetic markers for rapid M. tuberculosis resistance to INH and/or RIF detection directly in clinical specimens or in clinical isolates. Therefore, in the present work, the frequency of mutations at codons 315 and 463 of the katG gene and at the 69-bp region of the rpoB gene of M. tuberculosis clinical isolates resistant to INH and/or RIF, respectively, from northeast Mexico were determined.
MATERIALS AND METHODS M. tuberculosis isolates A total of 48 INH- and RIF-resistant clinical isolates, 19 INHresistant isolates, and 9 RIF-resistant isolates were randomly selected and provided over a 1-year period (1998) from the State Laboratory of the Department of Health of Nuevo León, Guadalupe, Mexico (60 isolates) and from the Regional Center of Infectious Disease Control of the School of Medicine of the Autonomous University of Nuevo León, Monterrey, Mexico (16 isolates). All of the isolates were from sputum samples limited to one per patient and were previously characterized by conventional biochemical tests13 and by their mycolic acid patterns.3–5,10 Furthermore, the resistance phenotype were previously determined by the indirect proportion method,6 following the recommendations of the National Committee for Clinical Laboratory Standards,19 and the mycolic acid index method.27,28 M. tuberculosisH37Ra (ATCC 25177) was used as susceptible control strain in both genetic analyses.
Isolation of DNA Mycobacteria were harvested from Lowenstein-Jensen slants in 3 ml of TE (10 mM Tris-HCl, 1 mM EDTA pH 8.0), thermally inactivated at 80°C for 1 hr and centrifuged (13,000 3 g for 5 min). Genomic DNA were isolated from mycobacterial cells lysed by enzymatic means.2 DNA solutions were quantified by densitometry of a stained ethidium bromide (2 mg/ml) 1% agarose electrophoresis gel using: a mini-single cell 1-1408 (FOTODYNE, Hartland, WI), 90 volts for 45 min, a DNA from Escherichia coli with a high degree of purity as standard, the Gel Doc 1000 (Bio-Rad, Hercules, CA) instrument, and the program Molecular Analyst 1.5 (Bio-Rad, Hercules, CA).
Detection of mutations at codons 315 and 463 of the katG gene To detect mutations at codons 315 and 463 of the katG gene in DNA samples from the 67 INH-resistant clinical isolates by
PCR-RFLP, an oligonucleotide pair was designed [59-CGGTCACACTTTCGGTAAGAC-3 9 (katG-I) and 59-CGGAGTTGAATGACTCCTGG-3 9 (katG-D)], using the sequence of the gene katG from GenBank (National Institutes of Health, accession number g488439), Primer3,21 Oligo 4.0 (Molecular Biology Insights Inc., Cascade, CO, USA), Amplify 1.0,9 DNA Strider 1.115 programs, and the BLAST service of the National Institutes of Health via Internet.1 The designed primers were DMT-off synthesized in an Oligo 1000 automated DNA synthesizer (Beckman Inc., Fullerton, CA), quantified by UV absorption at 260 nm, dissolved in sterile high-performance liquid chromatography (HPLC)-grade water adjusting the concentration to 5 mM, and then were used in the PCR assays without prior purification. PCR reactions were performed in an MJ Research thermal cycler (Watertown, MA) using standard amplification conditions: 25 ml of reaction volume, containing PCR buffer (Promega, Madison, WI), 1.5 mM MgCl2, 6 ng of genomic DNA/ml, 200 mM dNTPs, 0.5 mM of each primer, sterile water, and 1.25 U Taq DNA polymerase (Promega, Madison, WI). A 30-cycle amplification program was used: 94°C for 1 min, 61°C for 30 sec, and 72°C for 1 min, with a first denaturation step of 94°C for 5 min and a final elongation step of 72°C for 5 min. To detect mutations at codon 315, digestions of the amplified products with MspA1I (New England BioLabs, Beverly, MA) were carried out in a 15-ml reaction volume containing 1.5 ml of 103 buffer (NEBuffer 4, New England BioLabs, Beverly, MA), 2.0 ml of amplified product, 1.5 ml of 100 mg/ml bovine serum albumin (BSA) solution (New England BioLabs, Beverly, MA), sterile water, and 1 U of restriction enzyme. In addition, to detect mutations at codon 463, digestions of the amplified products with NciI (GibcoBRL, Gaithersburg, MD) were carried out in a 15-ml reaction volume containing 1.5 ml of 103 buffer (GibcoBRL, Gaithersburg, MD), 2.0 ml of amplified product, sterile water, and 1 U of restriction enzyme. Both reactions were carried out at 37°C for 3 hr and later 5 ml of the reaction mixture was analyzed in a 2.5% agarose electrophoresis gel stained with ethidium bromide (2 mg/ml) using a mini-single cell (FOTODYNE, Hartland, WI), 100 volts for 60–70 min.
Detection of mutations at the 69-bp region of the rpoB gene All of the DNA samples from the 57 RIF-resistant clinical isolates were analyzed using the INNO-LiPA RifTB kit according to the manufacturer’s instructions and as has been described.20 The line probe assay kit was kindly provided by Murrex Diagnostics Inc. (Norcross, GA).
Statistical analysis The mutation frequencies in the katG and rpoB genes were statistically compared with those reported from other countries using a Chi-square-test; p , 0.05 was significant.
RESULTS The designed PCR primers rendered a 786-bp fragment that contains codons 315 and 463 of katG. Figure 1 shows two agarose gels with the band patterns of the digested amplified
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katG/rpoB MUTATIONS IN INH-/RIF-RESISTANT ISOLATES
FIG. 1. Restriction analysis of katG amplification products. (Numbers on both sides are in base pairs.) Both agarose gels. Left lane M, molecular size marker (AluI digest of pBR322); lanes AP, undigested M. tuberculosis H37Ra amplification product; right lane M, molecular size marker (Hae III digest of pUC18). For left agarose gel: lane Ra, MspA1I digest of M. tuberculosis H37Ra amplification product; lanes 1–6, several MspA1I digests of INH-resistant clinical isolate amplification products. For right agarose gel: lane Ra, NciI digest of M. tuberculosis H37Ra amplification product; lanes 1–6, several NciI digests of INH-resistant clinical isolate amplification products.
product with the enzymes MspA1I (left) and NciI (right). The third lane of both gels is the band pattern from the control strain (343 and 306 bp). On the left, lanes 3, 5, and 6 show the same band pattern as the band pattern of the control strain, indicating that these samples did not present the mutation at codon 315. Lanes 1, 2, and 4 show a different band pattern (409 and 306 bp) from the band pattern of the control strain, indicating that these samples presented a mutation at codon 315 of katG. On the right, lanes 1–6 show the same band pattern as the band pattern of the control strain, indicating that these samples did not present the mutation at codon 463 of katG. Of the 67 INHresistant clinical isolates tested, 36 (53.7%) showed the mutation at codon 315; however none of them showed the mutation at codon 463 of katG. Figure 2 shows eight of the hybridization strip patterns obtained. The first pattern corresponds to the wild-type pattern where all the S-type bands (S1, S2, S3, S4, and S5) were observed in the middle part of the strip. The second pattern corresponds to mutation S531L, where the absence of S5 band and the presence of the R5 band were observed. The third and fourth patterns correspond to mutations H526Y and H526D, respectively, where the absence of the S4 band and the presence of the R4a and R4b bands, respectively, were observed. The fifth pattern corresponds to mutation D516V, where the absence of the S2 band and the presence of the R2 band were observed. In the sixth, seventh, and eighth patterns, the absence of the S2, S3, and S4 bands, respectively, were observed, indicating the presence of a mutation in that specific DNA region. The specific probe for the M. tuberculosis complex nucleotide sequences included on each strip of the line probe kit was always positive for each of the 57 RIF-resistant isolates and for the
control strain. The majority of the RIF-resistant samples analyzed (49 of 57, 86.0%) reacted positively with one of the four R-type probes (R2, R4a, R4b, and R5) and always were accompanied with a negative reaction on the corresponding S probe (S2, S4, S4, and S5, respectively), except when dealing with mixed cultures (6 isolates). The R5 pattern (S531L mutation) was the most frequently observed (31 of 57, 54.4%), followed by R4a pattern (H526Y mutation) 13 isolates (22.8%), R4b pattern (H526D mutation) 4 isolates (7.0%), and R2 pattern (D516V mutation) 1 isolate (1.8%). The majority of the R5-type isolates were also INH-resistant isolates (27 of 31, 87.1%) without any appreciable correlation with the presence of the mutation at codon 315 of katG (12 with mutation, 15 without mutation). Seven of the 57 (12.3%) RIF-resistant isolates yielded hybridization patterns with one negative signal with an S probe in which the precise mutation could not be established (DS2 1 isolate, 1.8%; DS3 3 isolates, 5.3%; and DS4 3 isolates, 5.3%). No correlation between the types of rpoB gene mutations and the percentage of RIF-resistant colonies obtained by the indirect proportion method or the mycolic acid index value were observed. Overall, there was agreement between the line probe kit and phenotypic RIF-susceptibility test for 56 (98.2%) of 57 RIF-resistant isolates tested.
DISCUSSION The resistance of M. tuberculosis to INH and/or RIF is on the increase, and laboratory methods used to identify M. tuberculosis drug resistance currently require from weeks to months for results. This emphasizes the need for fast and reli-
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FIG. 2. Representative hybridization patterns for detecting rpoB mutations. Conj. Contr., Conjugate control; strip 1, wild-type pattern; strip 2, R5 pattern (S531L mutation); strip 3, R4a pattern (H526Y mutation); strip 4, R4b pattern (H526D mutation); strip 5, R2 pattern (D516V mutation); strip 6, DS2 pattern; strip 7, DS3 pattern; strip 8, DS4 pattern.
able methods for the detection of M. tuberculosis and the determination of its drug susceptibility both for optimal patient treatment and for control of the disease. The PCR-RFLP assay and the line probe kit used in this study may help meet this need. Both assays are able to detect and identify M. tuberculosis complex strains and simultaneously provide information with respect to the susceptibility of the strain to INH or RIF, respectively. In addition, both drugs are key elements in the treatment of tuberculosis. The basic principle of the PCR-RFLP assay is that a mutation at codon 315 or 463 of katG results in a restriction site deletion. The oligonucleotides and PCR conditions described here render an intense amplified product band with which it is easy to differentiate between the characteristic digestion pattern of M. tuberculosis strains with and those without the mutation at codon 315 and 463. In addition, these oligonucleotides are theoretically specific (via BLAST) for M. tuberculosis and M. bovis. This technique may be very useful for a routine mycobacterial laboratory that only needs to know the presence or absence of mutation at codon 315 of katG to infer the resistance to INH of a clinical isolate. Comparing the frequency of mutations at codons 315 and 463 of katG found with those reported elsewhere,11,17,18 significant differences (p , 0.05) were observed for most of them, although the mutation at codon 315 was the most frequently observed in all the cases. The absence of mutation at codon 463 of katG in the analyzed samples is in agreement with the findings of Shim et al.,22 Haas et al.,11 and Nachamkin et al.,18 who suggested that the mutation at this codon may be a polymorphism not related with resistance to INH. Knowing that the 67 analyzed samples for mutations at
codons 315 and 463 of katG were INH-resistant, the resistance to INH of the 31 samples that did not show either of the two mutations analyzed could be due to another mutation in the katG gene, keeping in mind that there are 22 allelic variations reported for this gene,17 or could be due to mutations in other genes as has been described by other authors.24 Although highly specific, the sensitivity of the PCR-RFLP assay for the codon 315 of katG was not sufficient to replace conventional methods for susceptibility testing. On the other hand, the principle of the line probe kit is that each nucleotide change in the 69bp region of rpoB should impede the hybridization of the amplified product and the corresponding wild-type probes. Of the 57 RIF-resistant isolates tested, only one sample (1.8%) showed a wild-type hybridization pattern, indicating that some RIF-resistant clinical isolates present a different resistance mechanism than that involved with point mutations in the 69-bp region of the rpoB gene. Comparing the rpoB mutation frequencies found here with those reported from other countries,7,12,16,20,29 significant differences were obtained (p , 0.05) for 38 of the 70 compared pairs. In all the cases, the R5 mutation was the most frequent. In the three U.S. populations7,12,29 and in our population, the R5, R4a, and R4b mutations were the most frequent (all together more of the 67% of mutations), whereas in Greece16 the R4a mutation has not been found; in Belgium-Romania, Asia, and Africa,20 the frequency reported for that mutation was lower than the American frequencies. On the other hand, a high S5 mutation frequency has been reported in Greece (17.6%) and Asia (16.1%), although we have not found this mutation in our population; a low frequency for that mutation has been reported in the three U.S. populations and in Belgium-
katG/rpoB MUTATIONS IN INH-/RIF-RESISTANT ISOLATES Romania population. This is in accordance with the results reported by Matsiota-Bernard et al.,16 who proposed a geographic distribution of M. tuberculosis strains with various rpoB mutations involved in resistance to RIF. Our results essentially confirm the work of other investigators who have found that the results of molecular analysis of rpoB correlate quite well with those of conventional susceptibility testing. The results obtained in this work indicate that the mutation detection at codon 315 of katG could be used as a screening assay prior to standard susceptibility testing, whereas mutations in the rpoB gene could be used successfully as genetic markers to detect rapidly RIF-resistant M. tuberculosis clinical isolates from northeast Mexico. The mutation analysis at codon 315 of katG gene will be positive for around half of the INHresistant clinical isolate cases and the point mutation analysis at the 69-pb region of rpoB gene using the line probe kit will be positive in more than 98% of RIF-resistant clinical isolate cases.
ACKNOWLEDGMENTS We are thankful for grant SA113-98 (PAICYT) from the Universidad Autónoma de Nuevo León for this work. We also thank the Hospital Universitario José Eleuterio González, Monterrey, México, and Laboratorio Estatal de Salud, Guadalupe, Mexico, for providing samples; Timothy W. Murray from Murrex Diagnostics Inc. (Norcross, GA, USA) for providing the INNO-LiPA Rif TB kits; Elvira Garza-González and María de la Luz Acevedo-Duarte for technical support; and R.M. ChandlerBurns for his critical reading of our manuscript. CMLA thanks the Consejo Nacional de Cienca y Tecnología (CONACYT) for her fellowship.
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