Mutations in putative mutator genes of Mycobacterium tuberculosis strains of the W-Beijing family. (Research).Alterations in genes involved in the repair of DNA DNA: see nucleic acid.
or deoxyribonucleic acid
One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. mutations (mut genes) result in an increased mutation frequency and better adaptability of the bacterium to stressful conditions. W-Beijing genotype strains displayed unique missense mis·sense
A section within a strand of messenger RNA containing a codon altered through mutation so that it codes for a different amino acid. alterations in three putative mut genes, including two of the mutT type (Rv3908 and mutT2) and ogt. These polymorphisms were found to be characteristic and unique to W-Beijing phylogenetic phy·lo·ge·net·ic
1. Of or relating to phylogeny or phylogenetics.
2. Relating to or based on evolutionary development or history. lineage. Analysis of the mut genes in 55 representative W-Beijing isolates suggests a sequential acquisition of the mutations, elucidating a plausible pathway of the molecular evolution of this clonal family. The acquisition of mut genes may explain in part the ability of the isolates of W-Beijing type to rapidly adapt to their environment.
Tuberculosis (TB) and AIDS cause more deaths in adults worldwide than any other infectious disease Infectious disease
A pathological condition spread among biological species. Infectious diseases, although varied in their effects, are always associated with viruses, bacteria, fungi, protozoa, multicellular parasites and aberrant proteins known as prions. . Globally, the number of TB cases is growing at a rate of 2% per year. Resistance, especially multidrug-resistance (MDR MDR,
n See multidrug resistance.
n the abbreviation for minimum daily requirement, specifically the Minimum Daily Requirements for Specific Nutrients compiled by the United States Food and Drug Administration. ), is an increasing problem (1) and a growing hazard to human health. Many outbreaks of MDR-TB MDR-TB Multi-Drug Resistant Tuberculosis , defined as resistance to at least rifampicin rifampicin /rif·am·pi·cin/ (rif´am-pi-sin) rifampin.
a derivative of rifamycin; an antibacterial and antifungal agent used in the treatment of mycobacterial infections, actinomycosis and histoplasmosis. and isoniazid isoniazid (ī'sōnī`əzĭd), drug used to treat tuberculosis. Also known as isonicotinic acid hydrazide, isoniazid is the most effective antituberculosis drug currently available. , have been reported, with poor response to therapy and very high disease and death rates. Some TB outbreaks have involved patients with HIV HIV (Human Immunodeficiency Virus), either of two closely related retroviruses that invade T-helper lymphocytes and are responsible for AIDS. There are two types of HIV: HIV-1 and HIV-2. HIV-1 is responsible for the vast majority of AIDS in the United States. co-infection (2,3). Although in several instances, MDR outbreaks associated with a particular genotype, such as the W strain, have been identified (4,5), drug-susceptible variants of the W strain account for most of this group of isolates characterized to date.
In 1995, the largest proportion of the Mycobacterium tuberculosis Mycobacterium tuberculosis
Mycobacterium tuberculosis strains from Beijing, China, shared a high degree of similarity in IS6110 restriction fragment length polymorphism restriction fragment length polymorphism
n. Abbr. RFLP
Intraspecies variations in the length of DNA fragments generated by the action of restriction enzymes and caused by mutations that alter the sites at which these enzymes act, changing (RFLP RFLP
restriction fragment length polymorphism
restriction fragment length polymorphism.
RFLP ) patterns and identical spoligo patterns (6). Subsequent molecular analyses have indicated that the W and Beijing isolates constitute a single group of strains designated as the W-Beijing genotype (Figure 1). The global distribution and success of M. tuberculosis M. tuberculosis,
n the bacterium responsible for tuberculosis, generally a respiratory infection in man; nonrespiratory tuberculosis is considered an indicator disease for AIDS. See also tuberculosis. isolates of the W-Beijing genotype have led to the hypothesis that these strains may have selective advantages over other M. tuberculosis strains. In addition to the W-MDR strain identified in New York City New York City: see New York, city.
New York City
City (pop., 2000: 8,008,278), southeastern New York, at the mouth of the Hudson River. The largest city in the U.S. , and areas in Cuba, Estonia, Vietnam, and Russia, the W-Beijing genotype has been significantly associated with drug resistance (7 and unpub. data). Several studies have suggested that the W-Beijing genotype strains are disseminating throughout the world (7). In Vietnam, the proportion of W-Beijing strains was 71% in patients <25 years of age and 41% for those >25 years of age (8). Furthermore, W-Beijing strains have been implicated im·pli·cate
tr.v. im·pli·cat·ed, im·pli·cat·ing, im·pli·cates
1. To involve or connect intimately or incriminatingly: evidence that implicates others in the plot.
2. in several TB epidemics globally, including ones in New York, Texas New York is a hamlet in Henderson County, Texas, USA, about 11 miles east of Athens. Geography
New York lies at the intersection of FM 804 and FM 607 in a stereotypically flat portion of East Texas, surrounded mostly by farm land. , California, South Carolina South Carolina, state of the SE United States. It is bordered by North Carolina (N), the Atlantic Ocean (SE), and Georgia (SW). Facts and Figures
Area, 31,055 sq mi (80,432 sq km). Pop. (2000) 4,012,012, a 15. , and New Jersey in the United States (9) and South Africa, Russia, and Spain (10). A recent study showed that 82% of MDR strains isolated in a prison in Azerbaijan, Eastern Europe, are of the W-Beijing genotype (11).
[FIGURE 1 OMITTED]
Ongoing research is focused on identifying the factors responsible for the worldwide spread of the W-Beijing strains and their ability to adapt and enhance their pathogenicity or virulence. Identifying a possible mechanism for increased adaptation of these bacteria to the human immunologic host defense system or human interventions such as anti-TB treatment is of the utmost importance. Such mechanisms may indicate how the bacterium adapts to the host, a prerequisite for an enhanced accumulation of genomic mutations associated with resistance. In M. tuberculosis, resistance to antibiotics occurs because of genomic mutations in certain genes, such as the katG gene for isoniazid (INH INH
Isotamine (CA), PMS Isoniazid (CA)
Pharmacologic class: Isonicotinic acid hydrazide
Therapeutic class: Antitubercular
) resistance and the rpoB gene for rifampicin resistance (12). In contrast to several other pathogens with MDR phenotypes, plasmid or transposon-mediated mechanisms of resistance have not been reported in M. tuberculosis (13-15). Since resistance to bacteriostatic bacteriostatic /bac·te·rio·stat·ic/ (bak-ter?e-o-stat´ik) inhibiting growth or multiplication of bacteria; an agent that so acts. in M. tuberculosis is exclusively due to genomic mutations, the bacterium would benefit from an increased mutation rate.
Recent studies provided evidence for a role of mutator A mutator may refer to:
A genus of gram-negative, nonsporeforming, rod-shaped bacteria. Motile species possess polar flagella. They are strictly aerobic, but some members do respire anaerobically in the presence of nitrate. isolates (16). Such phenotypes not only enable the bacteria to acquire resistance to antibiotics more easily but also facilitate their adaptation to a new niche. Bacteria can escape immune surveillance immune surveillance
See immunological surveillance. by modulating bacterial resistance to host defense mechanisms (16-18). This finding prompted us to investigate whether a similar situation exists in M. tuberculosis. We have undertaken a comprehensive comparative sequence analysis of selected target genes to evaluate and study the presence of mutations in putative genes expected to play a role in the mutation frequency in such strains.
Mutated phenotypes commonly result from defects in DNA repair (19). An in silico analysis suggested that most mismatch repair systems (e.g., mutS, mutL, or mutH) were missing in the M. tuberculosis genome (20). However, the frequency of spontaneous mutations in M. tuberculosis (in vitro in vitro /in vi·tro/ (in ve´tro) [L.] within a glass; observable in a test tube; in an artificial environment.
In an artificial environment outside a living organism. cultures) is similar to that found in other bacteria-carrying mismatch repair systems (21), which suggests that other DNA repair mechanisms must be present. Hypothetical open reading frames (ORF), similar to genes known to be responsible for the avoidance or repair of DNA lesions resulting from the alkylation alkylation /al·kyl·a·tion/ (al?ki-la´shun) the substitution of an alkyl group for an active hydrogen atom in an organic compound.
n. or oxidation of nucleotides, are present in the genome of M. tuberculosis. We searched for variations in these genes in 139 clinical isolates to detect possible mutations that could allow an enhanced adaptability to the host and increased resistance to anti-TB drugs.
We searched for mut genes variation in 139 M. tuberculosis complex strains originating from 35 different countries. Ninety-four of these strains were selected because they were representative strains characterized with 13 different genetic markers in previous studies (6,22).
This set comprised 125 M. tuberculosis strains, 1 M. africanum, 8 M. bovis, 3 M. bovis BCG BCG bacille Calmette-Guérin.
1. bacillus Calmette-Guérin
n.pr See bacille Calmette-Guórin. , and 2 M. microti. Fifty-five strains had a W-Beijing genotype; 12 had an MDR phenotype. Strains representing different branches of the W-Beijing genotype were studied. Eight MDR M. tuberculosis strains with a genotype other than Beijing were included. Five M. tuberculosis strains of the W-Beijing genotype and three strains of unrelated genotype were obtained from the national program for surveillance of MDR tuberculosis in Spain. Four M. tuberculosis W-Beijing genotype strains isolated in the Netherlands and one from Vietnam were included because they showed spoligo patterns with fewer than nine spacers. Five other W-Beijing genotype strains showed hybridization hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun)
1. crossbreeding; the act or process of producing hybrids.
2. molecular hybridization
3. to an additional spacer, as demonstrated by using the extended set of spacers, two of which lacked hybridization to spacer 37. Strain W4 is part of a drug-susceptible outbreak in New Jersey (4); W147 is a drug-resistant isolate widely spread in Russia (23). Eleven strains were representative of ancestral W-Beijing strains, which diverged early in the evolution of the W-Beijing phylogenetic lineage. Finally, 29 strains of another frequently observed genotype, the Haarlem genotype (6), were investigated.
The collection consisted of 55 W-Beijing genotype isolates, 29 Haarlem genotype isolates, 8 strains of the African genotype, 1 M. bovis strain, and 46 representatives of other genotypes. Principal genetic grouping (PGG PGG Purple, Green, and Gold (Mardi Gras beads colors)
PGG Project Galactic Guide
PGG Patrol Gunboat (Missile)
PGG Pendleton Grain Growers, Inc. (Pendleton, OR, USA) ), according to the polymorphism in katG and gyrA, was known (24) for most of the isolates in this study. Seventy-four strains belong to PGG 1, 54 to PGG 2, and 3 to PGG 3. All isolates were subjected to at least IS6110 RFLP typing and spoligotyping (6). Drug susceptibility was determined for 41 of 139 strains (Table 1 and 2). Several putative mut genes were annotated as such in the released genome sequence of M. tuberculosis (25). In addition, using the BLAST program (26), we identified Rv3908 as an ORF carrying a mutT domain (27) and have since named it mutT4.
Primers were designed to amplify putative mut genes: mutY (5'-CCGGCGACGAATCGCTCGTT-3', 5'-AGCTGGGACAGTCGTCGCGG-3'), mutM (5'-CTGGTTCGATGGTGATGACC-5', 5'-GTGCGCTCGACCCACAG-3'), mutT2 (5'-TCCGGATGATGATTTACCTCC-3', 5'-TCCGCCGGGTCGGGGAC-3'), mutT1 (5'-ATCGTCGGCGTGCCGTG-3', 5'-GTCAGCGTCCTGCCCGG-3'), mutT4 (5'-TCGAAGGTGGGCAA- ATCGTG-3' 5'-TGGGGTTCGCTGGAAGTGG-3'), ogt (5'-CAGCGCTCGCTGGCGCC-3', 5'-GACTCAG CCGCTCGCGA-3'), and mut T3 (5'-GTCACGTCTGTTAGGACCTC-3', 5'-CGCGCAACGGCTGCCGG-3'). Similar primers were designed to amplify the rpoB gene (5'-TACGGTCGGCGAGCTGATCC-3', 5'- TACGG- CGTTTCGATGAACC-3').
DNA sequencing was performed directly on the amplified fragments by using the dideoxy chain-termination method with the Big Dye Terminator Cycle sequencing Kit (Perkin Elmer Applied Biosystems, Courtaboeuf, France) on a GeneAmp polymerase chain reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is (PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) ) system 9600 (Perkin Elmer) and run on a DNA analysis DNA analysis Any technique used to analyze genes and DNA. See Chromosome walking, DNA fingerprinting, Footprinting, In situ hybridization, Jeffries' probe, Jumping libraries, PCR, RFLP analysis, Southern blot hybridization. system model 373 or 3100 (Applied Biosystems). Sequences of mutY, mutT2, mutT4, rpoB, mutT1, mutT3, and ogt of the M. tuberculosis strains H37Rv, CDC See Control Data, century date change and Back Orifice.
CDC - Control Data Corporation 1551, and MT210 were obtained from published sequences or at the TIGR TIGR The Institute for Genomic Research
TIGR Treasury Investment Growth Receipt
TIGR This Is Getting Ridiculous
TIGR Thermally Induced Gallium Removal
TIGR TSPI Interface for GPS/RAJPO Web site (available from: URL URL
in full Uniform Resource Locator
Address of a resource on the Internet. The resource can be any type of file stored on a server, such as a Web page, a text file, a graphics file, or an application program. : http://www.tigr.org/).
We searched for allele allele (əlēl`): see genetics.
Any one of two or more alternative forms of a gene that may occur alternatively at a given site on a chromosome. variation in putative genes coding for DNA repair enzymes: mutT (which hydrolyzes 8-oxo-deoxyguanosine triphosphate triphosphate /tri·phos·phate/ (tri-fos´fat) a salt containing three phosphate radicals.
A salt or ester containing three phosphate groups. ) (28), ogt (which removes methyl groups from O6-methylguanine in DNA) (29), mutM (formamidopyrimidine-DNA glycosylate) (30), and mutY (specific adenine adenine (ăd`ənĭn, –nīn, –nēn), organic base of the purine family. Adenine combines with the sugar ribose to form adenosine, which in turn can be bonded with from one to three phosphoric acid units, yielding the three glycosylate) (31) in 12 MDR M. tuberculosis strains. Subsequently, we genotyped for the observed single nucleotide polymorphism Noun 1. single nucleotide polymorphism - (genetics) genetic variation in a DNA sequence that occurs when a single nucleotide in a genome is altered; SNPs are usually considered to be point mutations that have been evolutionarily successful enough to recur in a (SNP SNP Scottish National Party
Noun 1. SNP - (genetics) genetic variation in a DNA sequence that occurs when a single nucleotide in a genome is altered; SNPs are usually considered to be point mutations that have been evolutionarily ) variation in 124 strains, members of the M. tuberculosis complex, and in the three published sequences of M. tuberculosis strains. In total, the sampling comprises 55 W-Beijing genotype M. tuberculosis strains, including 11 ancestral W-Beijing isolates (unpub. data), 84 M. tuberculosis strains of other genotypes, and 1 M. bovis strain.
Several putative mut genes were annotated as such in the released genome sequence of M. tuberculosis. A BLAST search using the E. coli E. coli: see Escherichia coli.
in full Escherichia coli
Species of bacterium that inhabits the stomach and intestines. E. coli can be transmitted by water, milk, food, or flies and other insects. mutT sequences as template identified, in addition to mutT1, mutT2, mutT3, the hypothetical ORF Rv3908, which we have designated as mutT4. The best matches with E. coli mutT gene were observed for mutT2 and mutT4. Figure 2 depicts sequence alignment of the conserved region of the different genes of the M. tuberculosis genome showing similarity with mutT of E. coli. The search for sequences similar to ogt, mutM, and mutY identified a single ORF in each case. Primers were designed for PCR amplification of all the genes mentioned above.
[FIGURE 2 OMITTED]
We first determined the sequences of the different genes mentioned above in 12 MDR M. tuberculosis strains (ZA20, ZA65, ZA67, ZA68, ZA69, ZA11, ZA16, ZA12, ZA13, ZAA ZAA Zeeman Atomic Absorption
ZAA Zone of Advanced Achievement 14, ZA17, and ZA19), including 5 W-Beijing strains (ZA20, ZA65, ZA67, ZA78, and ZA69). For the mutY, mutM, mutT1, and mutT3 putative genes, PCR amplification was obtained in all strains tested, but sequence analysis did not indicate any nucleotide changes at these loci except for the same silent SNP in mutT3 in strains with a Haarlem genotype. We confirmed these findings by sequencing mutT1, mutT3, mutM, and mutY in a collection of 26 MDR strains from North Africa. No variation was observed in mutT1 or mutM. Only one strain had a major variation in mutY. All Haarlem strains carried one characteristic silent mutation in mutT3 and one characteristic mutation in ogt (Ser 15 replaced by Thr). These defining SNPs were also observed for all Haarlem strains of this study. No other variations were observed in mutT1, mutT3, mutM, or mutY. However, comparative sequence analysis of H37Rv, CDC1551, and the five MDR-W-Beijing isolates indicated polymorphisms in mutT2, mutT4, and ogt. These mutations in mutT4, mutT2, and ogt were also found in the W-Beijing strain 210 (TIGR) but not in MDR strains other than those belonging to the W-Beijing genotype. We therefore decided to extend this investigation and look for mutations in these three genes in a collection of M. tuberculosis complex isolates, including well-defined branches of the W-Beijing phylogenetic lineage (Table 1 and 2).
In 43 of 55 strains with a W-Beijing genotype, either susceptible to bacteriostat bac·te·ri·o·stat
An agent, such as a chemical, that inhibits bacterial growth.
bac·teri·o·stat or MDR, we found a mutation in mutT4. Codon codon: see nucleic acid. 48 (CGG CGG Compagnie Generale de Geophysique
CGG Canadian Grenadier Guards (Canadian reserve military unit)
CGG Cancer Genetics Group (Birmingham, UK) ) of the annotated ORF had been changed to GGG GGG German Goo Girls (pornography website)
GGG Giggle (email, USENET, chat slang)
GGG Gadolinium Gallium Garnet
GGG Gimme Gimme Gimme (TV show) , resulting in the amino acid amino acid (əmē`nō), any one of a class of simple organic compounds containing carbon, hydrogen, oxygen, nitrogen, and in certain cases sulfur. These compounds are the building blocks of proteins. substitution of Arg by Gly (Table 1 and 2). All 11 W-Beijing isolates known to be closely related to the ancestral W-Beijing strain (AM, HI, N16, DU2, DV, LB2, KY, IK, 122(C11), 113, and 107(LB)) were found to have the wild-type genotype as all other 84 isolates with a genotype other than W-Beijing.
Thirty-nine of 43 W-Beijing strains with the mutation in mutT4 carried an additional mutation in mutT2 and in ogt. The mutT2 mutation constitutes a change in codon 58 (GGA GGA Generalized Gradient Approximation
GGA Good Game All
ggA Geschützte Geographische Angabe (German: Protected Geographical Indication)
GGA Global Gecko Association
GGA Georgia Geocachers Association to CGA (Color/Graphics Adapter) The first video display standard for the IBM PC. This low-resolution system was superseded by EGA and then VGA. CGA required a digital RGB Color Display monitor. See PC display modes.
CGA - Color Graphics Adapter ), resulting in an amino acid substitution of Gly by Arg. The active site of the E. coli MutT enzyme comprises amino acids 53, 56, 57, and 98. Therefore, a mutation Gly to Arg at position 58 may have a important effect on enzyme activity Enzyme activity
A measure of the ability of an enzyme to catalyze a specific reaction.
Mentioned in: Glucose-6-Phosphate Dehydrogenase Deficiency and lead to a mutator phenotype.
All 39 W-Beijing isolates carrying the mutT2 polymorphism at codon 58 also displayed a concurrent silent mutation in codon 12 (Gly GGG to GGA Gly) of the ogt gene. Of four possible codons encoding for glycine glycine (glī`sēn), organic compound, one of the 20 amino acids commonly found in animal proteins. Glycine is the only one of these amino acids that is not optically active, i.e. , GGG and GGA had the lowest relative synonymous codon usage Relative Synonymous Codon Usage (RSCU) is a simple measure of non-uniform usage of synonymous codons in a coding sequence. RSCU values are the number of times a particular codon is observed, relative to the number of times that the codon would be observed for a uniform synonymous (RSCU RSCU Rescue ) in genes with high expression levels (0.20 and 0.17, respectively, compared to 1.32 and 2.31 for GGU GGU Golden Gate University
GGU Grønlands Geologiske Undersøgelse (Greenland's Geological Survey)
GGU Gold's Gym of Utah
GGU Gas Generator Unit
GGU Government Guarantee and Undertaking (Vietnam) and GGC GGC Girl Guides of Canada
GGC Greenwood Genetic Center (South Carolina)
GGC Gwasanaeth Gwaed Cymru (Welsh Blood Service)
GGC Generalized Goppa Code
GGC Grosvenor Gallery Company ). For genes with low expression levels, the RSCU values are 0.92, 0.37, 0.65, and 2.06 for GGG, GGA, GGU, and GGC, respectively (32).
The five W-Beijing isolates with a mutation in mutT4 and a wild-type mutT2 gene did not contain the ogt silent mutation on codon 12 either. Instead, they all shared a dinucleotide dinucleotide /di·nu·cleo·tide/ (di-nldbomack´le-o-tid?) one of the cleavage products into which a polynucleotide may be split, itself composed of two mononucleotides.
n. substitution in codon 37 (ACC See adaptive cruise control. to CTC CTC - Cornell Theory Center ) of ogt, resulting in amino acid substitution of Arg to Leu Leu leucine.
leucine. . These five W-Beijing isolates of 43 with the mutT4 mutations, without the mutT2 (codon 58) or the ogt (codon 12) mutations, differed molecularly from all other W-Beijing isolates in their spoligotype pattern and accompanying deletion flanking the DR locus. Four of five were isolated from Dutch patients in the Netherlands; the fifth originated from a patient in Vietnam. The Vietnamese isolate (no. 94) shared >95% IS6110 pattern similarity with the Dutch isolate 115 when standard RFLP analysis was used. Overall, the five isolates were closely related to each other according to IS6110 profiling (>90% similarity). Spacer 37 in the DR locus of Dutch isolates 114 and 139 was absent, while sample 115 was missing spacers 37 and 38, and 111 had a deletion of spacers 38 and 39 but not spacer 37, suggesting that these isolates may belong to a different sublineage. A tentative phylogeny of the W-Beijing strains analyzed in this study is proposed in Figure 3. Seven of nine MDR W-Beijing strains carried missense mutations in two muT genes (mutT2 and mutT4), and two had a missense mutation in both mutT4 and ogt (Table 1 and 2).
[FIGURE 3 OMITTED]
No mutations in mutT4 or in mutT2 were observed in any of the 84 M. tuberculosis complex strains, including 19 strains of PGG1, 54 strains of PGG2, and 2 strains of PGG3; the strains originated from 29 countries and were a genotype other than W-Beijing. A Thr15Ser mutation was observed in 24 of 29 strains of the Harlem family. No other change was observed in ogt.
Resistance to rifampicin in MDR strains was correlated with mutations in the rpoB gene. The three tested MDR W-Beijing strains isolated in Spain, with the mutations at the mutT2 and mutT4 loci, harbored a different mutation in the rpoB gene. These strains were isolated from patients who had emigrated from Eastern Europe to Spain (ZA67, ZA68, and ZA69). Analysis of the IS6110 RFLP of the respective isolates showed a difference of a single band. These findings suggest that the three strains may be related. The acquisition of the three different mutations in the rpoB gene leading to rifampicin resistance must have occurred after the acquisition of mutations in the putative nucleotide repair enzyme genes mutT4 and mutT2.
Our results show that M. tuberculosis strains of the W-Beijing genotype acquired missense mutations in DNA repair genes. These M. tuberculosis W-Beijing genotype strains are genetically highly conserved and widespread. DNA repair genes have been previously shown to be associated with mutator phenotypes in other microorganisms. The success of this group of strains may result in part from mutations in DNA repair enzymes, which might provided a true selective advantage for these bacteria to adapt and persist, including through the acquisition of resistance to anti-TB drugs. Mutations in the DNA repair genes might be the evolutionary answer of the TB bacillus bacillus (bəsĭl`əs), any rod-shaped bacterium or, more particularly, a rod-shaped bacterium of the genus Bacillus. Some bacterium in the genus cause disease, for example B. to increase adaptation to hosts. This adaptation will lead to increasing trends in the TB epidemic in the coming decades. The World Health Organization considers MDR and resistance as a problem of local rather than of global importance (1). If the relative contribution of W-Beijing genotype strains to the current worldwide TB epidemic is increasing as suggested (7), this approach should be revised. In areas with an increasing problem with MDR-TB, such as Estonia and Russia, W-Beijing genotype strains are predominantly associated with MDR cases (33). In Germany, the relative proportion of W-Beijing strains among isolates from resistant cases has increased from 12% in 1995 to 35% in 2000 (unpub. data). The latter observation may indicate an increasing influence of W-Beijing strains on the worldwide TB epidemic.
We identify polymorphisms in M. tuberculosis in genes that might result in a mutator phenotype and therefore a plausibly better adaptation of the bacilli bacilli /ba·cil·li/ (bah-sil´i) plural of bacillus.
see bacillus. to a hostile environment (34). Forty-three of 55 W-Beijing isolates analyzed were found to have a unique mutation on the ORF Rv3908. This ORF contains a Mutt domain and is denoted here as mutT4. Thirty-nine of 43 W-Beijing strains carried an additional and identical mutation in a second putative gene of the mutT family, mutT2, and an identical silent mutation in ogt.
The W-Beijing phylogenetic lineage probably acquired the mutation on codon 48 of the mutT4 only once and before other mutations associated with the mutator genes we describe. This mutation clearly distinguishes ancestral W-Beijing isolates from contemporary W-Beijing strains. The 11 W-Beijing isolates that did not have the characteristic mutT4 mutation on codon 48, consist of a collection of isolates known to be ancestral within this phylogenetic lineage, as determined by various other molecular techniques (unpub. data).
Nine of W-Beijing strains with the wild-type mutT2 gene had a characteristic mutation on codon 37 of the ogt gene, which suggests that these isolates constitute a branch of the W-Beijing family that diverged after the acquisition of the mutT4 mutation but before the development of the nucleotide substitution on mutT2. One strain carries the mutation 37 in ogt but no mutation in mutT4, a reversion that might have occurred after a transient mutator phenotype.
A mutation in mutT2 was always associated with a mutation in muT4. A first mutation may have occurred in mutT4 and thereafter a second mutation either in mutT2 or ogt was acquired. As observed for other bacterial populations, mutator phenotypes may be transient in many cases to limit deleterious effects (35). Identifying these mutations may aid in the identification of mut genes in M. tuberculosis. These mutations associated with mutator genes provide a reliable tool for the identification of W-Beijing isolates and thus a useful marker for strains endowed with capacity to yield epidemics. The biologic consequences of these mutations and function of these DNA repair genes are currently been investigated in the laboratory.
Nine MDR strains with a W-Beijing genotype were among strains carrying two missense mutations in putative mutator genes. Phylogenetically phy·lo·ge·net·ic
1. Of or relating to phylogeny or phylogenetics.
2. Relating to or based on evolutionary development or history: a phylogenetic classification of species. unrelated M. tuberculosis MDR isolates had no mutations within the DNA repair genes investigated in this study. Our data support the idea that M. tuberculosis strains of the W-Beijing genotype may have adapted to hostile environments, including exposure to anti-TB drugs, because of a succession of alterations of DNA repair enzymes. Other genes involved in other DNA repair mechanisms or in the fidelity of DNA replication may also be involved and remain to be investigated.
The acquisition of mutator alleles was described as an adaptive response of bacteria to a succession of different environments (18,35,36). After infecting a host, M. tuberculosis has to adapt to different environments such as alveolar macrophages and dendritic cells and subsequently to granuloma granuloma /gran·u·lo·ma/ (gran?u-lo´mah) pl. granulomas, granulo´mata an imprecise term for (1) any small nodular delimited aggregation of mononuclear inflammatory cells, or (2) such a collection of modified macrophages containing inactivated inactivated
rendered inactive; the activity is destroyed.
treated so that they are no longer able to produce evidence of growth or damaging effect on tissue. macrophages Macrophages
White blood cells whose job is to destroy invading microorganisms. Listeria monocytogenes avoids being killed and can multiply within the macrophage. or to activated macrophages after induction of the acquired immune responses. In addition, the bacilli have to adapt to the caseous caseous /ca·se·ous/ (ka´se-us) resembling cheese or curd; cheesy.
Of, relating to, or having the gross and microscopic features of tissue affected by caseation. media with low oxygen concentration in the center of tubercles and to different types of tissues during dissemination of the disease. Such variable growth conditions might select for mutations in M. tuberculosis strains, as described in other bacterial populations exposed to different environmental challenges. Mutations and selection might occur with an increased frequency caused by the toxic radicals produced in phagocytic cells Phagocytic cells
A cell that ingests microorganisms and foreign particles.
Mentioned in: Chronic Granulomatous Disease .
However, a mutator phenotype is often transient. Otherwise a continual accumulation of mutations would lead to deleterious effects and loss of fitness. No difference in the frequency of spontaneous mutations, resulting in a rifampin rifampin (rĭfăm`pĭn), antibiotic used in the treatment of tuberculosis. It is also used to eliminate the meningococcus microorganism from carriers and to treat leprosy, or Hansen's disease. resistance phenotype, was observed for W-Beijing strains (37). We suggest that a transient mutator phenotype allowed a better adaptation of W-Beijing strains. Subsequent compensatory mutations occurred to reverse the mutator phenotype. An alternative hypothesis alternative hypothesis Epidemiology A hypothesis to be adopted if a null hypothesis proves implausible, where exposure is linked to disease. See Hypothesis testing. Cf Null hypothesis. would be the existence of a higher mutation rate in specific conditions (i.e., in mutagenic mutagenic
inducing genetic mutation. radicals inside phagocytes). The accumulation of mutations leading to antibiotic resistance antibiotic resistance,
n the ability of certain strains of microorganisms to develop resistance to antibiotics.
antibiotic resistance in W-Beijing strains may be a consequence of the appearance of strains with a better adaptation to the hosts. MDR strains would be easily selected when patients with strains that have adapted better received inadequate anti-TB regimens.
Table 1. Characteristics of Mycobacterium tuberculosis complex strains originating from 35 different countries No. Country of Strains Genotype isolates isolation Group ZA20/65 W-Beijing 2 Spain 1 ZA67-69 W-Beijing 3 Spain 1 ZA11/16 Haarlem 2 Spain 2 ZA12-14/17 other 4 Spain nd ZA19 M. bovis 1 Spain ZA15 other 1 Spain nd ZA60-62 W-Beijing 3 Spain 1 CDC1551 1 USA 2 H37Rv 1 USA 3 MT210 W-Beijing 1 USA 1 20 W-Beijing 1 Mongolia 1 30 W-Beijing 1 South Africa 1 34 W-Beijing 1 Malaysia 1 43 W-Beijing 1 China 1 44 W-Beijing 1 Thailand 1 45 W-Beijing 1 Malaysia 1 91/102-6 W-Beijing 6 Vietnam 1 110/116/119/12 W-Beijing 8 the Netherlands 1 4-5/140-2 133 W-Beijing 1 South Africa 1 W4/10/126/129 W-Beijing 4 USA 1 W99 W-Beijing 1 Singapore 1 W147 W-Beijing 1 Russia 1 94 W-Beijing 1 Vietnam 1 111 W-Beijing 1 South Korea 1 115 W-Beijing 1 the Netherlands 1 5107(HG1) W-Beijing 1 USA 1 114, 139 W-Beijing 1 the Netherlands 1 166(HD6) W-Beijing 1 USA 1 165(001) W-Beijing 1 USA 1 107(LB) W-Beijing 1 USA 1 113 W-Beijing 1 the Netherlands 1 122(CI1) W-Beijing 1 USA 1 IK/KY/LB2/DV W-Beijing 6 Russia 1 /DU2/HI N16 W-Beijing 1 USA 1 AM W-Beijing 1 USA 1 Strains mutT2 mutT4 ZA20/65 wt Arg CGG 48 GGG Gly ZA67-69 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly ZA11/16 wt wt ZA12-14/17 wt wt ZA19 wt wt ZA15 wt wt ZA60-62 wt Arg CGG 48 GGG Gly CDC1551 wt wt H37Rv wt wt MT210 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly 20 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly 30 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly 34 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly 43 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly 44 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly 45 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly 91/102-6 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly 110/116/119/12 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly 4-5/140-2 133 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly W4/10/126/129 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly W99 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly W147 Gly GGA 58 CGA Arg Arg CGG 48 GGG Gly 94 wt Arg CGG 48 GGG Gly 111 wt Arg CGG 48 GGG Gly 115 wt Arg CGG 48 GGG Gly 5107(HG1) wt Arg CGG 48 GGG Gly 114, 139 wt Arg CGG 48 GGG Gly 166(HD6) wt Arg CGG 48 GGG Gly 165(001) wt wt 107(LB) wt wt 113 wt wt 122(CI1) wt wt IK/KY/LB2/DV wt wt /DU2/HI N16 wt wt AM wt wt Strains ogt ZA20/65 Arg CGC 37 Leu CTC ZA67-69 Gly GGG 12 Gly GGA ZA11/16 Thr ACC 15 Ser AGC ZA12-14/17 wt ZA19 wt ZA15 wt ZA60-62 Arg CGC 37 Leu CTC CDC1551 wt H37Rv wt MT210 Gly GGG 12 Gly GGA 20 Gly GGG 12 Gly GGA 30 Gly GGG 12 Gly GGA 34 Gly GGG 12 Gly GGA 43 Gly GGG 12 Gly GGA 44 Gly GGG 12 Gly GGA 45 Gly GGG 12 Gly GGA 91/102-6 Gly GGG 12 Gly GGA 110/116/119/12 Gly GGG 12 Gly GGA 4-5/140-2 133 Gly GGG 12 Gly GGA W4/10/126/129 Gly GGG 12 Gly GGA W99 Gly GGG 12 Gly GGA W147 Gly GGG 12 Gly GGA 94 Arg CGC 37 Leu CTC 111 Arg CGC 37 Leu CTC 115 Arg CGC 37 Leu CTC 5107(HG1) Arg CGC 37 Leu CTC 114, 139 wt 166(HD6) wt 165(001) Arg CGC 37 Leu CTC 107(LB) wt 113 wt 122(CI1) wt IK/KY/LB2/DV wt /DU2/HI N16 wt AM wt (a) wt, wild-type alleles (identical to H37Rv strain); nd, not determined Table 2. Characteristics of Mycobacterium tuberculosis complex strains originating from 35 different countries No. Country Strains Genotype isolates of isolation Group AU Haarlem 1 USA 2 3,5,22,32,39,48,50 Haarlem 10 Argentina 2 ,52-3,55 8 Haarlem 1 Vietnam 2 13/28 Haarlem 2 Sri Lanka 2 51 Haarlem 1 the Netherlands 2 57/59 Haarlem 2 Czech republic 2 84 Haarlem 1 Czech Republic 2 86/143/145 Haarlem 3 Bolivia 2 87 Haarlem 1 USA 2 99 Haarlem 1 Italy 2 123 Haarlem 1 Czech Republic 2 144/146-7 Haarlem 3 Bolivia 2 Apr-35 Africa 2 Rwanda 2 37 Africa 1 Uganda 2 40/120 Africa 2 Burundi 2 72 Africa 1 Central African 2 Republic 97 Africa 1 Uganda 2 121 Africa 1 Central African 2 Republic 2 BCG 1 the Netherlands 1 6/47/73/130 M. bovis 4 the Netherlands 1 12 Other 1 Tunisia 3 15/31 Other 2 Iran 2 16 Other 1 Canada 2 17 Other 1 Greenland 2 18 Other 1 USA 2 19/36/74 Other 2 India 1 25/62 M. microti 2 UK 1 26 Other 1 Zimbabwe 2 27 Other 1 Ethiopia 2 38/42 Other 2 Tahiti 2 41/46 Other 2 Chile 2 49 Other 1 Tanzania 1 56 Other 1 Curacao 2 64 Other 1 Honduras 2 65/112 Other 2 the Netherlands 1 71 BCG 1 Japan 1 76/101/126 M. bovis 3 Argentina 1 83 BCG 1 Russia 1 89/95 Other 2 Spain 2 96 Other 1 the Netherlands 3 98 Other 1 Ecuador 2 100 M. africanum 1 the Netherlands 1 108 Other 1 China 2 118 Other 1 Honduras 2 Strains mutT2 mut T4 ogt AU wt wt Thr ACC 15 Ser AGC 3,5,22,32,39,48,50 wt wt Thr ACC 15 Ser AGC ,52-3,55 8 wt wg Thr ACC 15 Ser AGC 13/28 wt wt Thr ACC 15 Ser AGC 51 wt wt Thr ACC 15 Ser AGC 57/59 wt wt wt 84 wt wt Thr ACC 15 Ser AGC 86/143/145 wt wt Thr ACC 15 Ser AGC 87 wt wt Thr ACC 15 Ser AGC 99 wt wt Thr ACC 15 Ser AGC 123 wt wt Thr ACC 15 Ser AGC 144/146-7 wt wt wt Apr-35 wt wt wt 37 wt wt wt 40/120 wt wt wt 72 wt wt wt 97 wt wt wt 121 wt wt wt 2 wt wt wt 6/47/73/130 wt wt wt 12 wt wt wt 15/31 wt wt wt 16 wt wt wt 17 wt wt wt 18 wt wt wt 19/36/74 wt wt wt 25/62 wt wt wt 26 wt wt wt 27 wt wt wt 38/42 wt wt wt 41/46 wt wt wt 49 wt wt wt 56 wt wt wt 64 wt wt wt 65/112 wt wt wt 71 wt wt wt 76/101/126 wt wt wt 83 wt wt wt 89/95 wt wt wt 96 wt wt wt 98 wt wt wt 100 wt wt wt 108 wt wt wt 118 wt wt wt (a) nd, not determined; wt, wild-type alleles (identical to H37Rv strain).
We thank Maxime Schwartz and Lluis Quintana-Murci for many helpful discussions and Xavier Nouvel for help with the manuscript.
This work received support from the European Commission (grant QLK2-CT-2000-00630), and from Louis D. French Academy of Sciences For the National Academy of Medicine, see .
The French Academy of Sciences (French: Académie des sciences) is a learned society, founded in 1666 by Louis XIV at the suggestion of Jean-Baptiste Colbert, to encourage and protect the spirit of French scientific award.
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Address for correspondence: Brigitte Gicquel, Unite de Genetique Mycobacterienne, Institut Pasteur, 28 rue du Dr. Roux Roux , Pierre Paul Émile 1853-1933.
French bacteriologist. His work with the diphtheria bacillus led to the development of antitoxins to neutralize pathogenic toxins. , 75015 Paris, France; fax: 33145688843; email: email@example.com
Mina Ebrahimi Rad, * Pablo Bifani, ([dagger]) Carlos Martin, ([double dagger]) Kristin Kremer, ([section]) Sofia Samper, ([double dagger]) Jean Rauzier, * Barry Kreiswirth, ([paragraph]) Jesus Blazquez, # Marc Jouan, * Dick van Soolingen, ([section]) and Brigitte Gicquel *
* Institut Pasteur, Paris, France; ([dagger]) Institut Pasteur, Lille, France; ([double dagger]) Universidad de Zaragoza, Zaragoza, Spain; ([section]) National Institute of Public Health and the Environment, Bilthoven, the Netherlands; ([paragraph]) Public Health Research Institute, Newark, New Jersey, USA; and # National Institute of Health, Madrid, Spain
Dr. Rad is a molecular biologist. Her expertise includes DNA amplification, sequencing, and cloning.