Drug Resistance Evolution of a Mycobacterium tuberculosis Strain from a Noncompliant Patient
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微生物临床杂志 2005年第7期
Dipartimento di Biologia Molecolare, Laboratorio di Microbiologia Molecolare e Biotecnologia, Universita di Siena, Siena
Dipartimento di Scienze Odontostomatologiche, Universita' di Cagliari, Cagliari
Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanita, 00161 Roma
Dipartimento di Microbiologia Clinica, Ospedali Riuniti, Ancona, Italy
ABSTRACT
The emergence and spread of multidrug-resistant (MDR) Mycobacterium tuberculosis (MT) represents a worldwide health care problem because of the difficulty in treating these infections. Development of drug resistance in MT arises mainly by mutation of chromosomal genes. To investigate the evolution of a MT population during a long-lasting infection, the phenotypic and genotypic changes in the drug resistance of 10 sequential MT isolates from a noncompliant chronically infected patient were investigated. During more than 12 years of active disease, a MDR population developed; molecular typing showed one single parental strain that infected the patient and persisted throughout the disease. Molecular analysis of the drug resistance-related genes revealed that discrete subpopulations evolved over time from the parental strain by acquiring and accumulating resistance-conferring mutations to isoniazid, rifampin, and streptomycin. Overall, these observations indicate that during a chronic infection, several subpopulations may coexist in the same patient with different drug susceptibility profiles.
INTRODUCTION
The emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis (MT) strains, i.e., strains resistant at least to isoniazid (INH) and rifampin (RMP), is alarming and represents a worldwide health care problem (40). In MT, drug resistance arises mainly by mutation of chromosomal genes, while the horizontal transfer of resistance-carrying genetic elements does not occur. Drug resistance acquisition by mutation is generally recognized as a one-step process in which bacteria become resistant by single point mutations. Because of this spontaneous phenomenon, a combined therapy is recommended to avoid the selection of resistant strains. A dramatic increase in the resistance and spread of MDR strains has been observed in recent years (40). These MDR strains carry multiple mutations in different resistance-related genes, and each mutation results from an independent mutational event. An MDR strain is thus the product of a multistep process, in which a progressive accumulation of genetic alterations occurs and results in the selection of a viable and fit bacterium. Drug-resistant bacteria are believed to grow more slowly than susceptible bacteria, since mutations conferring resistance reduce their overall fitness, a phenomenon known as cost of resistance (16). Several authors, however, have demonstrated that fit variants are quickly selected in a drug-resistant bacterial population, in which compensatory mutations eliminate the biological cost of resistance (1, 3, 17, 33).
All data reported on strain fitness generally refer to homogeneous bacterial populations, but due to continuously ongoing selective pressure in the host during infection, this might not be the case for a bacterial population infecting humans. The detailed characteristics of mycobacterial populations in the host during infection are still poorly understood. Kaplan et al. described the parallel evolution of different MT subpopulations from a unique strain in different sites of an infected lung (14). The simultaneous occurrence in an infected site of MT clones with different drug susceptibilities could affect the effectiveness of the therapy and the clinical outcome of the patient.
The observation that different resistant MT genotypes have arisen during 12 years of active tuberculosis (TB) in a single patient led us to investigate the evolution of a single MT population by studying the genotypic and phenotypic changes in the drug resistance during a chronic infection.
CASE REPORT
The patient studied was an Italian man with a history of intravenous drug abuse, in whom pulmonary TB was diagnosed when he was 29 years old. He presented at the Umberto I Hospital in Ancona, Italy, with fever, weakness, mild weight loss, cough, and diffuse cervical and axillar adenopathy. A chest radiograph showed pulmonary infiltrates in both upper lobes with extensive cavities; a Ziehl-Neelsen stain of a sputum smear revealed the presence of acid-fast bacilli. Further blood examinations discovered seropositivity to human immunodeficiency virus. On the basis of these findings and drug susceptibility testing (DST) showing susceptibility to all first-line anti-TB drugs, treatment including TB chemotherapy (INH, RMP, and ethambutol [EMB]) and zidovudine was begun. After an initial improvement, the patient became poorly compliant with the therapy and a 3-year follow-up control revealed many acid-fast bacilli in smears of gastric aspirate and sputum samples. The strain showed a simultaneous resistance to INH and RMP, i.e., it was an MDR strain. The patient refused to undergo further follow-up procedures, behaving in an extremely aggressive manner toward the health care providers. At 7 years after the onset of clinical symptoms, the patient was admitted into the hospital with a cytomegalovirus retinitis. His sputum was still positive for MT, and chemotherapy with EMB, rifabutin (RFB), pyrazinamide (PZA), and ciprofloxacin (CIP) was prescribed. The patient continued to be poorly compliant with the anti-TB treatment, and a new isolate, cultured the following year from sputum, was shown to be resistant to all first-line drugs. Finally, he died of a progressive wasting syndrome 12 years after his initial TB diagnosis. A necropsy was not done (Fig. 1).
MATERIALS AND METHODS
Mycobacterial isolation and DST. Ten sequential MT strains were isolated from sputum samples over 12 years of active disease. Respiratory specimens were liquefied and decontaminated by the N-acetyl-L-cysteine sodium hydroxide procedure (20) and cultured in Lwenstein-Jensen medium and by the radiometric BACTEC 460 TB system (Becton-Dickinson) (18, 31, 32, ). Microorganisms were identified as MT complex strains by DNA-RNA hybridization (Accuprobe; Gene Probe) (18, 20). All strains were grown in Middlebrook 7H9 medium (Difco) and stored at –80°C.
The DST on the clinical strains isolated was performed with the radiometric BACTEC 460 TB system (31, 32). MICs were determined with Middlebrook 7H11 agar (Difco) as previously described (7). Briefly, plates containing different drug concentrations were inoculated with approximately 2 x 102 and 2 x 103 CFU by a semiautomated inoculator (Multipoint Inoculator A400; Denley) and incubated at 37°C for 21 days. The following drugs were tested: INH, RMP, EMB, streptomycin (SM), RFB, CIP, ofloxacin, sparfloxacin, amikacin, kanamycin, capreomycin, cycloserine, para-aminosalicylic acid, thiacetazone, viomycin, and ethionamide. The MIC was defined as the lowest drug concentration inhibiting >99% of the inoculum. Susceptibility to PZA was determined by the BACTEC 460 TB method.
DNA preparation and molecular analysis. Genomic DNA from heat-inactivated cultures of MT was purified by the High Pure PCR Template Preparation kit (Roche Diagnostics), following the procedure for isolation of nucleic acids from bacteria or yeasts, according to the manufacturer's instructions. The analyzed genes were as follows: for RMP, the gene of the RNA polymerase beta-subunit (rpoB); for SM, the genes of ribosomal subunit S12 (rpsL) and 16S rRNA (rrs); for EMB, the putative arabinosyl transferase (embB) gene; and for INH, the catalase-peroxidase (katG) gene, the enoyl-reductase (inhA) gene, the alkyl hydroperoxidase C (ahpC) gene, the ahpC-oxyR intergenic region, the ferric uptake regulator homolog (furA) gene, and the iron-dependent regulator (ideR) gene. The direct automated sequencing of the PCR fragments containing the resistance-related genes was performed by standard procedures (11, 26). The sequences of both DNA strands were determined, each using as a template the product of a different PCR. Cycle sequencing reactions were performed with the Thermo Sequenase Fluorescent Labeled Primer Cycle Sequencing kit (Amersham Biosciences). As a sequencing template, 50 ng of crude PCR product and 2 pmol of each IRD800 infrared-labeled sequence primer (MWG-Biotech) were used. The cycle sequencing conditions were as follows: 1 cycle at 92°C for 2 min, and then 30 cycles at 60°C for 30 s, 72°C for 30 s, and 92°C for 30 s. The sequence products were analyzed on a LICOR 4000L automated infrared DNA sequencer apparatus (LI-COR Biosciences) (11, 26). The primers used in PCR and sequencing are listed in Table 2. Primers for rpoB and embB are described in references 13 and 26.
To detect mixed populations, fluorescence resonance energy transfer (FRET) probes for real-time PCR were designed as previously described (24). The probe sets were designed on the wild-type (wt) sequence; thus, the mutated allele showed a lower melting temperature (Tm) than the wild type (Fig. 2). Primers and probes are listed in Table 1. Real-time PCR was carried out with a LightCycler apparatus (Roche Diagnostics), and the mixture contained 1 μl of sample DNA, 10 pmol of primers, 2 pmol of FRET probes, 4 mM MgCl2, 1x Master Hybridization Probes reaction mixture (Roche Diagnostics), and PCR-grade sterile water to a final volume of 20 μl. Up to 32 samples were run in parallel by performing an initial 30-s denaturation step at 95°C, followed by 40 cycles of repeated denaturation (0 s at 95°C), annealing (10 s at 57°C), and polymerization (10 s at 72°C). The temperature transition rate was 20°C/s in all segments. In the final cycle, the melting curve was determined in the samples by initial heating to 95°C, cooling to 45°C, and subsequent controlled heating to 95°C with a temperature transition rate of 0.1°C/s. Primers and FRET probes used in real-time PCR are listed in Table 3.
The clinical isolates were genotyped by IS6110-based restriction fragment length polymorphism analysis to exclude the presence of different strains (7, 9).
Nucleotide sequence accession number. The sequence for the rpsL AAGACG K88T mutation described in this paper has been deposited to GenBank (accession no. AF398882).
RESULTS
Antimicrobial susceptibility. The MICs of the 10 MT isolates to 17 drugs are shown in Table 2. The MICs of INH, RMP, SM, and RFB increased during the 12 years of the patient's disease. In particular, the MICs of INH gradually increased from 0.06 to 2 μg/ml, with the strains P01 to P06 being susceptible and strains P07 to P10 resistant to this drug. In contrast, the MICs of SM, RMP, and RFB sharply increased, with strains P03 to P10 showing uniformly high values. Resistance to PZA also increased with time, with the strains from P01 to P07 being susceptible and the strains P08 to P10 being resistant. No increase over time in the MICs of EMB and in those of the other second-line drugs was seen. Data for MICs to second-line drugs are reported in Table 4.
Sequencing and real-time PCR. Sequencing analysis showed that all the 10 MT isolates carried the same katG GGGAGG G234R allele, suggesting that the same strain persisted over time. The DNA fingerprinting results on IS6110 confirmed that all sequential isolates belonged to a unique strain (data not shown) (7).
As to INH resistance, melting curve analysis after real-time PCR and nucleotide sequencing showed a wild-type katG gene in all isolates. However, a mutation in the position –9 (GA) of the intergenic region of the ahpC-oxyR pseudogene (numbering of intergenic region nucleotides is based on position relative to the mRNA start site, as identified in reference 41) was seen in strains P05 to P10. No other mutations were observed either in katG or in furA, inhA, and ideR. The strong increase in the resistance to RMP in strains P03 to P10 coincided with the presence of a TCGTTG S531L mutation in the rpoB gene of all these strains (codon numbering corresponds to the homologous gene in Escherichia coli; in MT, the codon is 450).
A more complex pattern was seen for the development of resistance to SM, as shown by the observation of a AAGACG K88T mutation in the rpsL gene of strain P03, followed by the appearance of heteroresistant populations containing the wild-type phenotype and K88T mutants (P04) and the wild-type phenotype and AAGAGG K43R mutants of the same gene (strains P05 to P07) (Fig. 2). The rpsL AAGACG K88T mutation is described in the present study for the first time; it was deposited in GenBank with the accession number AF398882. Strains P08, P09, and P10 contained only K43R mutants. No mutations were found in the rrs and embB genes. To confirm if the bacterial population, heteroresistant for streptomycin, was composed of a susceptible and a resistant subclone or of two resistant subclones (one of which was not detected by the probes), the proportion of resistant colonies was assayed. As reported in Table 5, only a fraction of the total bacterial population in samples P04, P05, and P07, which yielded heteroresistant genotypes, showed phenotypic resistance. In samples P02 and P08, in which no heteroresistance was detected, the phenotypic analysis matched the genotypic data.
DISCUSSION
Drug susceptibility testing of the 10 MT strains collected from the same patient throughout a 12-year course of TB revealed a progressive development of resistance to RMP, SM, INH, and PZA.
The emergence of the resistance to RMP in the third isolate coincided with the appearance of the rpoB S531L allele, the most frequently reported RMP resistance-conferring mutation (26, 37). This occurrence is in keeping with previous observations by our group showing that this mutation is the one most frequently found in Italy (26). The strain was also resistant to RFB, in accordance with the knowledge that mutations at codon rpoB 531 correlated with resistance to both compounds (4). In the third isolate, a high level of resistance against SM was also observed, in concomitance with the appearance of an AAGACG K88T mutation in the rpsL gene (Table 3). Previously described streptomycin resistance yielding mutations in this codon resulted in changes from lysine to arginine, but the simultaneous detection of this new substitution and the phenotypic increase of drug resistance correlates this "new" mutation to streptomycin resistance. This K88T mutant showed, probably, a lower fitness than that of the susceptible parental strain, as indicated by the fact that it was replaced over time by a mixture of the wild-type and AAGAGG K43R mutants and then by a pure K43R-containing population which persisted up to the death of the patient. Mutations in these two codons are known to be the most frequently associated with high-level SM resistance (5, 21, 35).
The INH resistance, detected from the P07 strain onwards, and the steady increase of MICs (strains P08 to P10) are not easily explained, as the only katG mutation identified (katG GGGAGG G234R) was present in all analyzed strains, even in the INH-susceptible strains (Table 3). Thus, the katG GGGAGG G234R allele may be considered a polymorphism and clearly does not affect the susceptibility to the drug. The mutation –9 GA in the intergenic region of oxyR-ahpC pseudogene also does not account for the emergence of INH resistance or for the increase in the MIC. Mutations in the promoter of ahpC have been demonstrated to induce INH resistance by activating the expression of AhpC peroxidase (10, 15, 30, 36). However, in our study the –9 GA mutation appeared in a still-sensitive strain (P05) and it was stably detected in all successive isolates, independently of the MIC (Table 3). This observation makes it difficult to impute the INH-resistant phenotype to the oxyR-ahpC –9 GA polymorphism. Complete sequencing of katG, furA, inhA, and ideR did not show any additional mutation. We thus could not track the MIC increase and resistance to INH of this strain to any of the mutations we detected. To explain the INH-resistant phenotype, the presence of mutations in other INH resistance-related genes, i.e., kasA (2, 19) or ndh (39), should be postulated.
Over the period of 12 years, mutations conferring drug resistance accumulated in these serial isolates of MT, possibly favored by the low level of patient compliance with therapy. The use of a molecular approach in drug resistance analysis allowed us to follow the evolution of the bacterial population over time. The detection of the katG GGGAGG G234R polymorphism in all isolates, confirmed by restriction fragment length polymorphism analysis results, demonstrates the persistence of a same strain in the patient throughout the period examined. Even though the drug susceptibility test first showed an increase in resistance and then an apparently stable phenotype, the molecular analysis of the MT strains revealed a dynamic process, characterized by a parallel evolution of discrete subpopulations, each carrying different drug resistance-conferring mutations. The initial pansusceptible population (P01 and P02) became resistant to RMP by acquiring the rpoB TCGTTG S531L mutation (Fig. 1; Table 3). From this mutated variant, two genotypes developed, the first one carrying the rpsL AAGACG K88T mutation and the second one having the oxyR-ahpC –9 GA substitution. A mixed population is in fact present in sample P04 in which, together with the rpsL AAGACG K88T mutated subpopulation, a second subpopulation exists that is the wild type for the rpsL gene (Fig. 1; Table 3). A similar situation was detected with sample P05, in which the whole population carried the oxyR-ahpC –9 GA mutation. However, two subpopulations were present, only one of which carried the rpsL AAGAGG K43R mutation (Fig. 1; Table 3). In all cases, the genotypic heteroresistance detected matched the phenotypic drug susceptibility testing data (Table 5).
The multiresistant strain isolated from sample P05 onward displaced all other clones and was the only detectable clone in three last samples. The emergence of this "successful" MDR strain was a multistep event, resulting from progressive accumulation of genetic alterations, possibly conferring a selective advantage for bacterial survival. The low compliance with therapy may have elicited the selection of resistant strains, which also persisted after the stop of treatment. This is consistent with the assumption that antibiotic-resistant bacteria adapt to eliminate the cost of resistance. Several studies demonstrated that drug-resistant bacteria with attenuated fitness accumulate compensatory mutations that restore fitness without altering the level of bacterial resistance (1, 3, 8, 17, 29, 33, 34). Overall, our data support the idea that in the lung of a chronically infected patient a single founder strain undergoes multiple events of mutagenesis, leading to the evolution of discrete subpopulations with different resistance profiles. These subpopulations are the result of a parallel evolution and changes in the relative prevalence of single subclones. Similar data have been described by other investigators analyzing heteroresistant mycobacterial strains (6, 23, 27, 28, 38).
The detection of mixed populations highlights the dynamic evolution of subpopulations from a single founder strain. At each moment during an infection, the bacterial population can be a mixture of different subclones, wild type or mutated for any marker; the different variants may became prevalent in diverse times of disease. The prevalence of each subpopulation could affect the outcome of DST and/or the effectiveness of therapy. Knowing the existence and prevalence of different subclones and their different drug susceptibilities could be useful for clinical management of the patient. Our data are consistent with recent studies in which different MT subpopulations were found in serial isolates recovered from MDR chronic TB infections and from various lesions of the same infected lung (14, 25). Taken together, these studies indicate that a systemic molecular search for bacterial subpopulations may be appropriate to ascertain their clinical impact.
ACKNOWLEDGMENTS
This work was supported in part by AIDS Project—Opportunistic Infections and TB grants 50D23 and 50F26 from the Italian Ministry of Health and from the FP5 Quality of Life program grant QLK2-CT-2002-01612 from the European Community.
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Dipartimento di Scienze Odontostomatologiche, Universita' di Cagliari, Cagliari
Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanita, 00161 Roma
Dipartimento di Microbiologia Clinica, Ospedali Riuniti, Ancona, Italy
ABSTRACT
The emergence and spread of multidrug-resistant (MDR) Mycobacterium tuberculosis (MT) represents a worldwide health care problem because of the difficulty in treating these infections. Development of drug resistance in MT arises mainly by mutation of chromosomal genes. To investigate the evolution of a MT population during a long-lasting infection, the phenotypic and genotypic changes in the drug resistance of 10 sequential MT isolates from a noncompliant chronically infected patient were investigated. During more than 12 years of active disease, a MDR population developed; molecular typing showed one single parental strain that infected the patient and persisted throughout the disease. Molecular analysis of the drug resistance-related genes revealed that discrete subpopulations evolved over time from the parental strain by acquiring and accumulating resistance-conferring mutations to isoniazid, rifampin, and streptomycin. Overall, these observations indicate that during a chronic infection, several subpopulations may coexist in the same patient with different drug susceptibility profiles.
INTRODUCTION
The emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis (MT) strains, i.e., strains resistant at least to isoniazid (INH) and rifampin (RMP), is alarming and represents a worldwide health care problem (40). In MT, drug resistance arises mainly by mutation of chromosomal genes, while the horizontal transfer of resistance-carrying genetic elements does not occur. Drug resistance acquisition by mutation is generally recognized as a one-step process in which bacteria become resistant by single point mutations. Because of this spontaneous phenomenon, a combined therapy is recommended to avoid the selection of resistant strains. A dramatic increase in the resistance and spread of MDR strains has been observed in recent years (40). These MDR strains carry multiple mutations in different resistance-related genes, and each mutation results from an independent mutational event. An MDR strain is thus the product of a multistep process, in which a progressive accumulation of genetic alterations occurs and results in the selection of a viable and fit bacterium. Drug-resistant bacteria are believed to grow more slowly than susceptible bacteria, since mutations conferring resistance reduce their overall fitness, a phenomenon known as cost of resistance (16). Several authors, however, have demonstrated that fit variants are quickly selected in a drug-resistant bacterial population, in which compensatory mutations eliminate the biological cost of resistance (1, 3, 17, 33).
All data reported on strain fitness generally refer to homogeneous bacterial populations, but due to continuously ongoing selective pressure in the host during infection, this might not be the case for a bacterial population infecting humans. The detailed characteristics of mycobacterial populations in the host during infection are still poorly understood. Kaplan et al. described the parallel evolution of different MT subpopulations from a unique strain in different sites of an infected lung (14). The simultaneous occurrence in an infected site of MT clones with different drug susceptibilities could affect the effectiveness of the therapy and the clinical outcome of the patient.
The observation that different resistant MT genotypes have arisen during 12 years of active tuberculosis (TB) in a single patient led us to investigate the evolution of a single MT population by studying the genotypic and phenotypic changes in the drug resistance during a chronic infection.
CASE REPORT
The patient studied was an Italian man with a history of intravenous drug abuse, in whom pulmonary TB was diagnosed when he was 29 years old. He presented at the Umberto I Hospital in Ancona, Italy, with fever, weakness, mild weight loss, cough, and diffuse cervical and axillar adenopathy. A chest radiograph showed pulmonary infiltrates in both upper lobes with extensive cavities; a Ziehl-Neelsen stain of a sputum smear revealed the presence of acid-fast bacilli. Further blood examinations discovered seropositivity to human immunodeficiency virus. On the basis of these findings and drug susceptibility testing (DST) showing susceptibility to all first-line anti-TB drugs, treatment including TB chemotherapy (INH, RMP, and ethambutol [EMB]) and zidovudine was begun. After an initial improvement, the patient became poorly compliant with the therapy and a 3-year follow-up control revealed many acid-fast bacilli in smears of gastric aspirate and sputum samples. The strain showed a simultaneous resistance to INH and RMP, i.e., it was an MDR strain. The patient refused to undergo further follow-up procedures, behaving in an extremely aggressive manner toward the health care providers. At 7 years after the onset of clinical symptoms, the patient was admitted into the hospital with a cytomegalovirus retinitis. His sputum was still positive for MT, and chemotherapy with EMB, rifabutin (RFB), pyrazinamide (PZA), and ciprofloxacin (CIP) was prescribed. The patient continued to be poorly compliant with the anti-TB treatment, and a new isolate, cultured the following year from sputum, was shown to be resistant to all first-line drugs. Finally, he died of a progressive wasting syndrome 12 years after his initial TB diagnosis. A necropsy was not done (Fig. 1).
MATERIALS AND METHODS
Mycobacterial isolation and DST. Ten sequential MT strains were isolated from sputum samples over 12 years of active disease. Respiratory specimens were liquefied and decontaminated by the N-acetyl-L-cysteine sodium hydroxide procedure (20) and cultured in Lwenstein-Jensen medium and by the radiometric BACTEC 460 TB system (Becton-Dickinson) (18, 31, 32, ). Microorganisms were identified as MT complex strains by DNA-RNA hybridization (Accuprobe; Gene Probe) (18, 20). All strains were grown in Middlebrook 7H9 medium (Difco) and stored at –80°C.
The DST on the clinical strains isolated was performed with the radiometric BACTEC 460 TB system (31, 32). MICs were determined with Middlebrook 7H11 agar (Difco) as previously described (7). Briefly, plates containing different drug concentrations were inoculated with approximately 2 x 102 and 2 x 103 CFU by a semiautomated inoculator (Multipoint Inoculator A400; Denley) and incubated at 37°C for 21 days. The following drugs were tested: INH, RMP, EMB, streptomycin (SM), RFB, CIP, ofloxacin, sparfloxacin, amikacin, kanamycin, capreomycin, cycloserine, para-aminosalicylic acid, thiacetazone, viomycin, and ethionamide. The MIC was defined as the lowest drug concentration inhibiting >99% of the inoculum. Susceptibility to PZA was determined by the BACTEC 460 TB method.
DNA preparation and molecular analysis. Genomic DNA from heat-inactivated cultures of MT was purified by the High Pure PCR Template Preparation kit (Roche Diagnostics), following the procedure for isolation of nucleic acids from bacteria or yeasts, according to the manufacturer's instructions. The analyzed genes were as follows: for RMP, the gene of the RNA polymerase beta-subunit (rpoB); for SM, the genes of ribosomal subunit S12 (rpsL) and 16S rRNA (rrs); for EMB, the putative arabinosyl transferase (embB) gene; and for INH, the catalase-peroxidase (katG) gene, the enoyl-reductase (inhA) gene, the alkyl hydroperoxidase C (ahpC) gene, the ahpC-oxyR intergenic region, the ferric uptake regulator homolog (furA) gene, and the iron-dependent regulator (ideR) gene. The direct automated sequencing of the PCR fragments containing the resistance-related genes was performed by standard procedures (11, 26). The sequences of both DNA strands were determined, each using as a template the product of a different PCR. Cycle sequencing reactions were performed with the Thermo Sequenase Fluorescent Labeled Primer Cycle Sequencing kit (Amersham Biosciences). As a sequencing template, 50 ng of crude PCR product and 2 pmol of each IRD800 infrared-labeled sequence primer (MWG-Biotech) were used. The cycle sequencing conditions were as follows: 1 cycle at 92°C for 2 min, and then 30 cycles at 60°C for 30 s, 72°C for 30 s, and 92°C for 30 s. The sequence products were analyzed on a LICOR 4000L automated infrared DNA sequencer apparatus (LI-COR Biosciences) (11, 26). The primers used in PCR and sequencing are listed in Table 2. Primers for rpoB and embB are described in references 13 and 26.
To detect mixed populations, fluorescence resonance energy transfer (FRET) probes for real-time PCR were designed as previously described (24). The probe sets were designed on the wild-type (wt) sequence; thus, the mutated allele showed a lower melting temperature (Tm) than the wild type (Fig. 2). Primers and probes are listed in Table 1. Real-time PCR was carried out with a LightCycler apparatus (Roche Diagnostics), and the mixture contained 1 μl of sample DNA, 10 pmol of primers, 2 pmol of FRET probes, 4 mM MgCl2, 1x Master Hybridization Probes reaction mixture (Roche Diagnostics), and PCR-grade sterile water to a final volume of 20 μl. Up to 32 samples were run in parallel by performing an initial 30-s denaturation step at 95°C, followed by 40 cycles of repeated denaturation (0 s at 95°C), annealing (10 s at 57°C), and polymerization (10 s at 72°C). The temperature transition rate was 20°C/s in all segments. In the final cycle, the melting curve was determined in the samples by initial heating to 95°C, cooling to 45°C, and subsequent controlled heating to 95°C with a temperature transition rate of 0.1°C/s. Primers and FRET probes used in real-time PCR are listed in Table 3.
The clinical isolates were genotyped by IS6110-based restriction fragment length polymorphism analysis to exclude the presence of different strains (7, 9).
Nucleotide sequence accession number. The sequence for the rpsL AAGACG K88T mutation described in this paper has been deposited to GenBank (accession no. AF398882).
RESULTS
Antimicrobial susceptibility. The MICs of the 10 MT isolates to 17 drugs are shown in Table 2. The MICs of INH, RMP, SM, and RFB increased during the 12 years of the patient's disease. In particular, the MICs of INH gradually increased from 0.06 to 2 μg/ml, with the strains P01 to P06 being susceptible and strains P07 to P10 resistant to this drug. In contrast, the MICs of SM, RMP, and RFB sharply increased, with strains P03 to P10 showing uniformly high values. Resistance to PZA also increased with time, with the strains from P01 to P07 being susceptible and the strains P08 to P10 being resistant. No increase over time in the MICs of EMB and in those of the other second-line drugs was seen. Data for MICs to second-line drugs are reported in Table 4.
Sequencing and real-time PCR. Sequencing analysis showed that all the 10 MT isolates carried the same katG GGGAGG G234R allele, suggesting that the same strain persisted over time. The DNA fingerprinting results on IS6110 confirmed that all sequential isolates belonged to a unique strain (data not shown) (7).
As to INH resistance, melting curve analysis after real-time PCR and nucleotide sequencing showed a wild-type katG gene in all isolates. However, a mutation in the position –9 (GA) of the intergenic region of the ahpC-oxyR pseudogene (numbering of intergenic region nucleotides is based on position relative to the mRNA start site, as identified in reference 41) was seen in strains P05 to P10. No other mutations were observed either in katG or in furA, inhA, and ideR. The strong increase in the resistance to RMP in strains P03 to P10 coincided with the presence of a TCGTTG S531L mutation in the rpoB gene of all these strains (codon numbering corresponds to the homologous gene in Escherichia coli; in MT, the codon is 450).
A more complex pattern was seen for the development of resistance to SM, as shown by the observation of a AAGACG K88T mutation in the rpsL gene of strain P03, followed by the appearance of heteroresistant populations containing the wild-type phenotype and K88T mutants (P04) and the wild-type phenotype and AAGAGG K43R mutants of the same gene (strains P05 to P07) (Fig. 2). The rpsL AAGACG K88T mutation is described in the present study for the first time; it was deposited in GenBank with the accession number AF398882. Strains P08, P09, and P10 contained only K43R mutants. No mutations were found in the rrs and embB genes. To confirm if the bacterial population, heteroresistant for streptomycin, was composed of a susceptible and a resistant subclone or of two resistant subclones (one of which was not detected by the probes), the proportion of resistant colonies was assayed. As reported in Table 5, only a fraction of the total bacterial population in samples P04, P05, and P07, which yielded heteroresistant genotypes, showed phenotypic resistance. In samples P02 and P08, in which no heteroresistance was detected, the phenotypic analysis matched the genotypic data.
DISCUSSION
Drug susceptibility testing of the 10 MT strains collected from the same patient throughout a 12-year course of TB revealed a progressive development of resistance to RMP, SM, INH, and PZA.
The emergence of the resistance to RMP in the third isolate coincided with the appearance of the rpoB S531L allele, the most frequently reported RMP resistance-conferring mutation (26, 37). This occurrence is in keeping with previous observations by our group showing that this mutation is the one most frequently found in Italy (26). The strain was also resistant to RFB, in accordance with the knowledge that mutations at codon rpoB 531 correlated with resistance to both compounds (4). In the third isolate, a high level of resistance against SM was also observed, in concomitance with the appearance of an AAGACG K88T mutation in the rpsL gene (Table 3). Previously described streptomycin resistance yielding mutations in this codon resulted in changes from lysine to arginine, but the simultaneous detection of this new substitution and the phenotypic increase of drug resistance correlates this "new" mutation to streptomycin resistance. This K88T mutant showed, probably, a lower fitness than that of the susceptible parental strain, as indicated by the fact that it was replaced over time by a mixture of the wild-type and AAGAGG K43R mutants and then by a pure K43R-containing population which persisted up to the death of the patient. Mutations in these two codons are known to be the most frequently associated with high-level SM resistance (5, 21, 35).
The INH resistance, detected from the P07 strain onwards, and the steady increase of MICs (strains P08 to P10) are not easily explained, as the only katG mutation identified (katG GGGAGG G234R) was present in all analyzed strains, even in the INH-susceptible strains (Table 3). Thus, the katG GGGAGG G234R allele may be considered a polymorphism and clearly does not affect the susceptibility to the drug. The mutation –9 GA in the intergenic region of oxyR-ahpC pseudogene also does not account for the emergence of INH resistance or for the increase in the MIC. Mutations in the promoter of ahpC have been demonstrated to induce INH resistance by activating the expression of AhpC peroxidase (10, 15, 30, 36). However, in our study the –9 GA mutation appeared in a still-sensitive strain (P05) and it was stably detected in all successive isolates, independently of the MIC (Table 3). This observation makes it difficult to impute the INH-resistant phenotype to the oxyR-ahpC –9 GA polymorphism. Complete sequencing of katG, furA, inhA, and ideR did not show any additional mutation. We thus could not track the MIC increase and resistance to INH of this strain to any of the mutations we detected. To explain the INH-resistant phenotype, the presence of mutations in other INH resistance-related genes, i.e., kasA (2, 19) or ndh (39), should be postulated.
Over the period of 12 years, mutations conferring drug resistance accumulated in these serial isolates of MT, possibly favored by the low level of patient compliance with therapy. The use of a molecular approach in drug resistance analysis allowed us to follow the evolution of the bacterial population over time. The detection of the katG GGGAGG G234R polymorphism in all isolates, confirmed by restriction fragment length polymorphism analysis results, demonstrates the persistence of a same strain in the patient throughout the period examined. Even though the drug susceptibility test first showed an increase in resistance and then an apparently stable phenotype, the molecular analysis of the MT strains revealed a dynamic process, characterized by a parallel evolution of discrete subpopulations, each carrying different drug resistance-conferring mutations. The initial pansusceptible population (P01 and P02) became resistant to RMP by acquiring the rpoB TCGTTG S531L mutation (Fig. 1; Table 3). From this mutated variant, two genotypes developed, the first one carrying the rpsL AAGACG K88T mutation and the second one having the oxyR-ahpC –9 GA substitution. A mixed population is in fact present in sample P04 in which, together with the rpsL AAGACG K88T mutated subpopulation, a second subpopulation exists that is the wild type for the rpsL gene (Fig. 1; Table 3). A similar situation was detected with sample P05, in which the whole population carried the oxyR-ahpC –9 GA mutation. However, two subpopulations were present, only one of which carried the rpsL AAGAGG K43R mutation (Fig. 1; Table 3). In all cases, the genotypic heteroresistance detected matched the phenotypic drug susceptibility testing data (Table 5).
The multiresistant strain isolated from sample P05 onward displaced all other clones and was the only detectable clone in three last samples. The emergence of this "successful" MDR strain was a multistep event, resulting from progressive accumulation of genetic alterations, possibly conferring a selective advantage for bacterial survival. The low compliance with therapy may have elicited the selection of resistant strains, which also persisted after the stop of treatment. This is consistent with the assumption that antibiotic-resistant bacteria adapt to eliminate the cost of resistance. Several studies demonstrated that drug-resistant bacteria with attenuated fitness accumulate compensatory mutations that restore fitness without altering the level of bacterial resistance (1, 3, 8, 17, 29, 33, 34). Overall, our data support the idea that in the lung of a chronically infected patient a single founder strain undergoes multiple events of mutagenesis, leading to the evolution of discrete subpopulations with different resistance profiles. These subpopulations are the result of a parallel evolution and changes in the relative prevalence of single subclones. Similar data have been described by other investigators analyzing heteroresistant mycobacterial strains (6, 23, 27, 28, 38).
The detection of mixed populations highlights the dynamic evolution of subpopulations from a single founder strain. At each moment during an infection, the bacterial population can be a mixture of different subclones, wild type or mutated for any marker; the different variants may became prevalent in diverse times of disease. The prevalence of each subpopulation could affect the outcome of DST and/or the effectiveness of therapy. Knowing the existence and prevalence of different subclones and their different drug susceptibilities could be useful for clinical management of the patient. Our data are consistent with recent studies in which different MT subpopulations were found in serial isolates recovered from MDR chronic TB infections and from various lesions of the same infected lung (14, 25). Taken together, these studies indicate that a systemic molecular search for bacterial subpopulations may be appropriate to ascertain their clinical impact.
ACKNOWLEDGMENTS
This work was supported in part by AIDS Project—Opportunistic Infections and TB grants 50D23 and 50F26 from the Italian Ministry of Health and from the FP5 Quality of Life program grant QLK2-CT-2002-01612 from the European Community.
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