Abstract
Genotypic analysis of 103 multidrug-resistant Mycobacterium tuberculosis strains isolated in Germany in 2001 revealed that mutations in codon 531 (75.7%) of the rpoB gene and codon 315 (88.4%) of the katG gene are most frequent. Beijing genotype strains (60.2% of all isolates) displayed a different distribution of resistance mutations than non-Beijing strains.
The molecular patterns of mutations conferring resistance to rifampin (RMP), mainly in the 81-bp hot spot region of the rpoB gene (5, 12, 21), and isoniazid (INH), mainly in katG, inhA, and oxyR-ahpC (9, 13, 16, 20), of Mycobacterium tuberculosis strains isolated in Germany in 1994-1995 and 1997 have been explored in previous investigations (3, 5, 17). Since that time, we observed a shift in the population structure of multidrug-resistant (MDR) tuberculosis strains, documented by a rising proportion of the Beijing genotype (7). In order to obtain recent data on drug-resistant strains circulating in Germany, in the present study we investigated resistance mutations of MDR strains isolated in 2001.
Genotypic analysis of RMP and INH resistance of 113 M. tuberculosis strains (103 MDR and 10 randomly chosen fully susceptible strains as controls) was carried out by use of real-time PCR and sequencing analysis (19). These samples represent more than 90% of all MDR cases reported in 2001 (7, 18).
In all 103 MDR isolates, mutations in the rpoB gene were detected, mostly in the 81-bp hot spot region. However, for one isolate a mutation could be detected only after cultivation on RMP-containing medium and reexamination. Fourteen different mutations in seven codons of the rpoB gene were found (Table 1). Codon 531 was most frequently affected in 78 of the 103 strains (75.7%). Other mutations were detected in rpoB 526 in 14 strains (13.6%) and in rpoB 516 in 3 strains (2.9%), and one was detected in codon 176, outside the hot spot region. One triple mutation, affecting codons 531 and 522 and involving a deletion of codon 519, was found. None of the 10 susceptible control strains carried a mutation in rpoB.
TABLE 1.
DNA sequencing and real-time PCR data for MDR M. tuberculosis strains from Germany, stratified for Beijing and non-Beijing strainsa
| MDR strain group (no. of strains) | Affected rpoB codon(s) | Nucleotide/amino acid change(s) | Affected katG, inhA, or ahpC codon | Nucleotide/amino acid change(s) | No. (%) of strains |
|---|---|---|---|---|---|
| All (103) | 531 | TCG→TTG/Ser→Leu | katG 315 | AGC→ACC/Ser→Thr | 64 (62.1) |
| TCG→TTG/Ser→Leu | None | 3 (2.9) | |||
| TCG→TTG/Ser→Leu | katG 315 | AGC→ACA/Ser→Thr | 2 (1.9) | ||
| TCG→TTG/Ser→Leu | katG 315 | AGC→AAC/Ser→Asn | 2 (1.9) | ||
| TCG→TTG/Ser→Leu | inhA 209 | C→T | 2 (1.9) | ||
| TCG→TTT/Ser→Phe | katG 315 | AGC→ACC/Ser→Thr | 2 (1.9) | ||
| TCG→TGG/Ser→Trp | katG 315 | AGC→ACC/Ser→Thr | 1 (1.0) | ||
| TCG→TGG/Ser→Trp | inhA 209 | C→T | 1 (1.0) | ||
| TCG→TGG/Ser→Trp | None | 1 (1.0) | |||
| 526 | CAC→AAC/His→Asn | katG 315 | AGC→ACC/Ser→Thr | 4 (3.9) | |
| CAC→CTC/His→Leu | katG 315 | AGC→ACC/Ser→Thr | 3 (2.9) | ||
| CAC→TAC/His→Tyr | katG 315 | AGC→ACC/Ser→Thr | 2 (1.9) | ||
| CAC→CGC/His→Arg | katG 315 | AGC→ACC/Ser→Thr | 2 (1.9) | ||
| CAC→CGC/His→Arg | ahpC-oxyR | C(−52)T | 1 (1.0) | ||
| CAC→GAC/His→Asp | katG 315 | AGC→ACC/Ser→Thr | 1 (1.0) | ||
| CAC→TGC/His→Cys | katG 315 | AGC→ACC/Ser→Thr | 1 (1.0) | ||
| 516 | GAC→GTC/Asp→Val | katG 315 | AGC→ACC/Ser→Thr | 2 (1.9) | |
| GAC→TAC/Asp→Tyr | katG 315 | AGC→ACC/Ser→Thr | 1 (1.0) | ||
| 522 | TCG→CAG/Ser→Gln | katG 315 | AGC→ACC/Ser→Thr | 1 (1.0) | |
| TCG→TTG/Ser→Leu | None | 1 (1.0) | |||
| 518 | AAC→ATC/Asn→Ile | None | 1 (1.0) | ||
| 513 | CAA→CCA/Gln→Pro | None | 1 (1.0) | ||
| 517 | Delb | katG 315 | AGC→ACC/Ser→Thr | 1 (1.0) | |
| 514-516 | Delb | katG 315 | AGC→ACC/Ser→Thr | 1 (1.0) | |
| 176 | GTC→TTC/Val→Phe | ahpC-oxyR | G(−48)A | 1 (1.0) | |
| 519/522/531 | Delb, TCG→TTG/Ser→Leu, and TCG→TTG/Ser→Leu | katG 315 | AGC→ACC/Ser→Thr | 1 (1.0) | |
| Beijing (62) | 531 | TCG→TTG/Ser→Leu | katG 315 | AGC→ACC/Ser→Thr | 48 (77.4) |
| TCG→TTG/Ser→Leu | inhA 209 | C→T | 1 (1.6) | ||
| TCG→TTG/Ser→Leu | None | 1 (1.6) | |||
| TCG→TTT/Ser→Phe | katG 315 | AGC→ACC/Ser→Thr | 2 (3.2) | ||
| 526 | CAC→AAC/His→Asn | katG 315 | AGC→ACC/Ser→Thr | 2 (3.2) | |
| CAC→CTC/His→Leu | katG 315 | AGC→ACC/Ser→Thr | 2 (3.2) | ||
| CAC→TAC/His→Tyr | katG 315 | AGC→ACC/Ser→Thr | 2 (3.2) | ||
| CAC→CGC/His→Arg | katG 315 | AGC→ACC/Ser→Thr | 1 (1.6) | ||
| 518 | AAC→ATC/Asn→Ile | None | 1 (1.6) | ||
| 516 | GAC→GTC/Asp→Val | katG 315 | AGC→ACC/Ser→Thr | 1 (1.6) | |
| 519/522/531 | Delb, TCG→TTG/Ser→Leu, and TCG→TTG/Ser→Leu | katG 315 | AGC→ACC/Ser→Thr | 1 (1.6) | |
| Non-Beijing (41) | 531 | TCG→TTG/Ser→Leu | katG 315 | AGC→ACC/Ser→Thr | 16 (39.0) |
| TCG→TTG/Ser→Leu | katG 315 | AGC→ACA/Ser→Thr | 2 (4.9) | ||
| TCG→TTG/Ser→Leu | katG 315 | AGC→AAC/Ser→Asn | 2 (4.9) | ||
| TCG→TTG/Ser→Leu | inhA 209 | C→T | 1 (2.4) | ||
| TCG→TTG/Ser→Leu | None | 2 (4.9) | |||
| TCG→TGG/Ser→Trp | katG 315 | AGC→ACC/Ser→Thr | 1 (2.4) | ||
| TCG→TGG/Ser→Trp | inhA 209 | C→T | 1 (2.4) | ||
| TCG→TGG/Ser→Trp | None | 1 (2.4) | |||
| 526 | CAC→AAC/His→Asn | katG 315 | AGC→ACC/Ser→Thr | 2 (4.9) | |
| CAC→GAC/His→Asp | katG 315 | AGC→ACC/Ser→Thr | 1 (2.4) | ||
| CAC→CGC/His→Arg | katG 315 | AGC→ACC/Ser→Thr | 1 (2.4) | ||
| CAC→CGC/His→Arg | ahpC-oxyR | C(−52)T | 1 (2.4) | ||
| CAC→TGC/His→Cys | katG 315 | AGC→ACC/Ser→Thr | 1 (2.4) | ||
| CAC→CTC/His→Leu | katG 315 | AGC→ACC/Ser→Thr | 1 (2.4) | ||
| 522 | TCG→CAG/Ser→Gln | katG 315 | AGC→ACC/Ser→Thr | 1 (2.4) | |
| TCG→TTG/Ser→Leu | None | 1 (2.4) | |||
| 516 | GAC→GTC/Asp→Val | katG 315 | AGC→ACC/Ser→Thr | 1 (2.4) | |
| GAC→TAC/Asp→Tyr | katG 315 | AGC→ACC/Ser→Thr | 1 (2.4) | ||
| 513 | CAA→CCA/Gln→Pro | None | 1 (2.4) | ||
| 517 | Delb | katG 315 | AGC→ACC/Ser→Thr | 1 (2.4) | |
| 514-516 | Delb | katG 315 | AGC→ACC/Ser→Thr | 1 (2.4) | |
| 176 | GTC→TTC/Val→Phe | ahpC-oxyR | G(−48)A | 1 (2.4) |
Concerning INH resistance, for 96 of the 103 MDR isolates (93.2%) a mutation in the genes analyzed was found (Table 1). Since no mutation was detected for the seven MDR strains (6.8%) after cultivation on INH-containing medium, presumably not heteroresistance but mutations in other regions of katG (16, 19) or genes not included in these investigation, such as kasA (10) or ndh (8), are the explanation for this finding. None of the susceptible strains carried a mutation in the regions investigated. In the majority of MDR isolates (84.5%; 87 of 103), a distinct nucleotide change in katG codon 315 from AGC (wild-type sequence) to ACC (S315T) was present. Four isolates (3.9%) carried other exchanges in codon 315 (two AAC and two ACA), three (2.9%) had mutations in the ribosome binding site region of inhA, and two (1.9%) had nucleotide exchanges in the regulatory region of the ahpC gene. This investigation showed a high prevalence of mutations in katG codon 315 (88.4%), which is contrary to results from previous studies performed in low-incidence countries (13) and even a study performed in Germany in 1994-1995 (44% katG substitutions) (3). Comparable high frequencies of the katG 315 mutations were found in northwestern Russia (93.6%) (11), in Latvia (91.0%) (22), and in Lithuania (85.7%) (2). With the additional information that the majority of patients, although residing in Germany at the time of strain isolation, originated from countries of the former Soviet Union (7), the high frequency of the katG mutations can probably be explained by an importation of strains from these regions. This conclusion was further supported when the distribution of resistance-conferring mutations was stratified for Beijing and non-Beijing strains. Of the 103 MDR isolates, 62 (60.2%) have been identified as Beijing genotype strains by IS6110 restriction fragment length polymorphism and their characteristic spoligotyping pattern (4, 6, 23).
Among the Beijing strains, a high rate of mutations was found in rpoB codon 531 (52 of 62 strains; 83.9%), whereas this portion was significantly lower within the 41 non-Beijing strains (26 of 41 strains; 63.4%; P = 0.02). In contrast, mutations in codon 526 were more frequent in non-Beijing strains (7 of 41 strains; 17.1%) than in Beijing strains (7 of 62 strains; 11.3%), but this difference was statistically not significant (P = 0.4). Concerning the distribution of mutations in the katG gene, the prevalence of the S315T mutation was significantly higher in the MDR Beijing group (59 of 62; 95.2%) than in the MDR non-Beijing group (28 of 41; 68.3%; P < 0.001). Furthermore, the MDR Beijing strains exhibited a great number of isolates (48 of 62; 77.4%) with an identical pattern of mutations (rpoB S531L and katG S315T) compared to only 16 of 41 isolates (39.0%) in the non-Beijing group (P < 0.001).
In conclusion, comparing MDR Beijing and non-Beijing genotype strains with respect to their mutations conferring RMP or INH resistance, a marked difference in the distribution of mutations was observed. Comparable differences have also been found for katG S315T mutations in a northwestern Russian setting (11). However, no association of specific mutations with a certain spoligotype pattern or genotype could be detected in recently published studies analyzing the prevalence of rpoB mutations in southeast Asia (15) or rpoB and katG mutations in Latvia (22) and England (1).
Since the proportion of Beijing genotype strains among MDR strains from Germany has changed markedly from 19.2% in 1995 to 58.3% in 2001 (7), this has also resulted in a shift of resistance mutations determined in MDR strains. Comparing the data from this study with the distribution of rpoB mutations present in RMP-resistant strains isolated in Germany found in previous studies, an increase of the mutations of rpoB codon 531 was assessed as follows: 1994-1995, 39% (17); 1997, 65% (5); and 2001, 75.7%. Accordingly, we observed a high rate of katG codon 315 mutations compared with the study of Dobner and colleagues (88.4 versus 44%) (3). To the best of our knowledge, this is the first study demonstrating the influence of strain importation on the prevalence of resistance mutations among strains in a given setting. In this context, the fact that the katG S315T mutation has no impact on the bacterial fitness (14) is of especial importance. Thus, the presence of particular clones of MDR strains might have a direct impact on transmission dynamics of MDR tuberculosis. As a consequence, the increased rate of strains carrying particular resistance mutations in line with the increasing proportion of Beijing strains may lead to a changed situation concerning transmission of MDR strains in Germany.
Acknowledgments
This work was supported by a grant from the Bundesministerium für Gesundheit und Soziale Sicherung, Berlin, Germany (325-4539-84/3.2).
We are grateful to Olfert Landt (TIB MOLBIOL Synthese Labor, Berlin, Germany) for designing and providing us with parts of the primers and FRET probes. We thank Kirsten Ott, Ilse Radzio, Merle Fischer, and Frauke Schaefer, Forschungszentrum Borstel, for excellent technical assistance.
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