Mass-spectrometry based minisequencing method for the rapid detection of drug resistance in Mycobacterium tuberculosis

A MALDI TOF MS based minisequencing method has been developed and applied for the analysis of rifampin (RIF)- and isoniazid (INH)-resistant M. tuberculosis strains. Eight genetic markers of RIF resistance-nucleotide polymorphisms located in RRDR of rpoB gene, and three of INH resistance including codon 315 of katG gene and − 8 and − 15 positions of the promoter region of fabG1-inhA operon were worked out. Based on the analysis of 100 M. tuberculosis strains collected from the Moscow region in 1997–2005 we deduced that 91% of RIF-resistant and 94% of INH-resistant strains can be identified using the technique suggested. The approach is rapid, reliable and allows to reveal the drug resistance of M. tuberculosis strains within 12 h after sample isolation.     

Whole Genome Sequencing Investigation of a Tuberculosis Outbreak in Port-au-Prince,Haiti Caused by a Strain with a “Low-Level” rpoB Mutation L511P – Insights into a Mechanism of Resistance Escalation

The World Health Organization recommends diagnosing Multidrug-Resistant Tuberculosis (MDR-TB) in high burden countries by detection of mutations in Rifampin (RIF) Resistance Determining Region of Mycobacterium tuberculosis rpoB gene with rapid molecular tests GeneXpert MTB/RIF and Hain MTBDRplus. Such mutations are found in >95% of Mycobacterium tuberculosis strains resistant to RIF by conventional culture-based drug susceptibility testing (DST). However routine diagnostic screening with molecular tests uncovered specific “low level” rpoB mutations conferring resistance to RIF below the critical concentration of 1 μg/ml in some phenotypically susceptible strains. Cases with discrepant phenotypic (susceptible) and genotypic (resistant) results for resistance to RIF account for at least 10% of resistant diagnoses by molecular tests and urgently require new guidelines to inform therapeutic decision making. Eight strains with a “low level” rpoB mutation L511P were isolated by GHESKIO laboratory between 2008 and 2012 from 6 HIV-negative and 2 HIV-positive patients during routine molecular testing. Five isolates with a single L511P mutation and two isolates with double mutation L511P&M515T had MICs for RIF between 0.125 and 0.5 μg/ml and tested susceptible in culture-based DST. The eighth isolate carried a double mutation L511P&D516C and was phenotypically resistant to RIF. All eight strains shared the same spoligotype SIT 53 commonly found in Haiti but classic epidemiological investigation failed to uncover direct contacts between the patients. Whole Genome Sequencing (WGS) revealed that L511P cluster isolates resulted from a clonal expansion of an ancestral strain resistant to Isoniazid and to a very low level of RIF. Under the selective pressure of RIF-based therapy the strain acquired mutation in the M306 codon of embB followed by secondary mutations in rpoB and escalation of resistance level. This scenario highlights the importance of subcritical resistance to RIF for both clinical management of patients and public health and provides support for introducing rpoB mutations as proxy for MICs into laboratory diagnosis of RIF resistance. This study illustrates that WGS is a promising multi-purpose genotyping tool for high-burden settings as it provides both “gold standard” sequencing results for prediction of drug susceptibility and a high-resolution data for epidemiological investigation in a single assay.     

Efflux Pump Gene Expression in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates

Isoniazid (INH) and rifampicin (RIF) are the two most effective drugs in tuberculosis therapy. Understanding the molecular mechanisms of resistance to these two drugs is essential to quickly diagnose multidrug-resistant (MDR) tuberculosis and extensive drug-resistant tuberculosis. Nine clinical Mycobacterium tuberculosis isolates resistant to only INH and RIF and 10 clinical pan-sensitive isolates were included to evaluate the expression of 20 putative drug efflux pump genes and sequence mutations in rpoB (RIF), katG (INH), the inhA promoter (INH), and oxyR-ahpC (INH). Nine and three MDR isolates were induced to overexpress efflux pump genes by INH and RIF, respectively. Eight and two efflux pump genes were induced to overexpress by INH and RIF in MDR isolates, respectively. drrA, drrB, efpA, jefA (Rv2459), mmr, Rv0849, Rv1634, and Rv1250 were overexpressed under INH or RIF stress. Most efflux pump genes were overexpressed under INH stress in a MDR isolates that carried the wild-type katG, inhA, and oxyR-ahpC associated with INH resistance than in those that carried mutations. The expression levels of 11 genes (efpA, Rv0849, Rv1250, P55 (Rv1410c), Rv1634, Rv2994, stp, Rv2459, pstB, drrA, and drrB) without drug inducement were significantly higher (P < 0.05) in nine MDR isolates than in 10 pan-sensitive isolates. In conclusion, efflux pumps may play an important role in INH acquired resistance in MDR M. tuberculosis, especially in those strains having no mutations in genes associated with INH resistance; basal expression levels of some efflux pump genes are higher in MDR isolates than in pan-sensitive isolates and the basal expressional differences may be helpful to diagnose and treat resistant tuberculosis.     

The Rationale for Using Rifabutin in the Treatment of MDR and XDR Tuberculosis Outbreaks

Genetically related Mycobacterium tuberculosis strains with alterations at codon 516 in the rpoB gene were observed amongst a substantial number of patients with drug resistant tuberculosis in the Eastern Cape Province (ECP) of South Africa. Mutations at codon 516 are usually associated with lower level rifampicin (RIF) resistance, while susceptibility to rifabutin (RFB) remains intact. This study was conducted to assess the rationale for using RFB as a substitution for RIF in the treatment of MDR and XDR tuberculosis outbreaks. Minimum inhibitory concentrations (MICs) of 34 drug resistant clinical isolates of M tuberculosis were determined by MGIT 960 and correlated with rpoB mutations. RFB MICs ranged from 0.125 to 0.25 µg/ml in the 34 test isolates thereby confirming phenotypic susceptibility as per critical concentration (CC) of 0.5 µg/ml. The corresponding RIF MICs ranged between 5 and 15 µg/ml, which is well above the CC of 1.0 µg/ml. Molecular-based drug susceptibility testing provides important pharmacogenetic insight by demonstrating a direct correlation between defined rpoB mutation and the level of RFB susceptibility. We suggest that isolates with marginally reduced susceptibility as compared to the epidemiological cut-off for wild-type strains (0.064 µg/ml), but lower than the current CC (≤0.5 µg/ml), are categorised as intermediate. Two breakpoints (0.064 µg/ml and 0.5 µg/ml) are recommended to distinguish between susceptible, intermediate and RFB resistant strains. This concept may assist clinicians and policy makers to make objective therapeutic decisions, especially in situations where therapeutic options are limited. The use of RFB in the ECP may improve therapeutic success and consequently minimise the risk of ongoing transmission of drug resistant M. tuberculosis strains.     

Acquisition of rifabutin resistance by a rifampicin resistant mutant of Mycobacterium tuberculosis involves an unusual spectrum of mutations and elevated frequency

Background

Mutations in a small region of the rpoB gene are responsible for most rifamycin resistance in Mycobacterium tuberculosis. In this study we have sequentially generated resistant strains to first rifampicin and then rifabutin. Portions of the rpoB gene were sequenced from 131 randomly selected mutants. Second round selection resulted in a changed frequency of specific mutations.

Methods

Mycobacterium tuberculosis (strain Mtb72) rifamycin resistant mutants were selected in vitro with either rifampicin or rifabutin. One mutant R190 (rpoB S522L) selected with rifampicin had a rifampicin MIC of 32 μg/ml but remained sensitive to rifabutin (MIC<0.8 μg/ml). This mutant was subjected to a second round of selection with rifabutin.

Results

All 105 first round resistant mutants derived from the parent strain (Mtb72) screened acquired mutations within the 81 bp rpoB hotspot. When the rifampicin resistant but rifabutin sensitive S522L mutant was subjected to a second round of selection, single additional rpoB mutations were identified in 24 (92%) of 26 second round mutants studied, but 14 (54%) of these strains contained mutations outside the 81 bp hotspot (codons 144, 146, 148, 505). Additionally, spontaneous rifabutin resistant mutants were produced at >10 times the frequency by the S522L mutant than the parent strain.

Conclusion

First round selection of mutation S522L with rifampicin increased the frequency and changed the spectrum of mutations identified after selection with rifabutin.     

The three faces of riboviral spontaneous mutation: spectrum, mode of genome replication, and mutation rate

Riboviruses (RNA viruses without DNA replication intermediates) are the most abundant pathogens infecting animals and plants. Only a few riboviral infections can be controlled with antiviral drugs, mainly because of the rapid appearance of resistance mutations. Little reliable information is available concerning i) kinds and relative frequencies of mutations (the mutational spectrum), ii) mode of genome replication and mutation accumulation, and iii) rates of spontaneous mutation. To illuminate these issues, we developed a model in vivo system based on phage Qß infecting its natural host, Escherichia coli. The Qß RT gene encoding the Read-Through protein was used as a mutation reporter. To reduce uncertainties in mutation frequencies due to selection, the experimental Qß populations were established after a single cycle of infection and selection against RT ⁻ mutants during phage growth was ameliorated by plasmid-based RT complementation in trans. The dynamics of Qß genome replication were confirmed to reflect the linear process of iterative copying (the stamping-machine mode). A total of 32 RT mutants were detected among 7,517 Qß isolates. Sequencing analysis of 45 RT mutations revealed a spectrum dominated by 39 transitions, plus 4 transversions and 2 indels. A clear template•primer mismatch bias was observed: A•C>C•A>U•G>G•U> transversion mismatches. The average mutation rate per base replication was ≈9.1×10⁻⁶ for base substitutions and ≈2.3×10⁻⁷ for indels. The estimated mutation rate per genome replication, μ_g, was ≈0.04 (or, per phage generation, ≈0.08), although secondary RT mutations arose during the growth of some RT mutants at a rate about 7-fold higher, signaling the possible impact of transitory bouts of hypermutation. These results are contrasted with those previously reported for other riboviruses to depict the current state of the art in riboviral mutagenesis.     

Probability Distribution of Drug-Resistant Mutants in Unselected Populations of Mycobacterium tuberculosis

The fluctuation test shows that Mycobacterium tuberculosis mutates to resistance to isoniazid, streptomycin, ethambutol and rifampin spontaneously and at random. The average mutation rates for the drugs, in the same order, were calculated to be 2.56 × 10⁻⁸, 2.95 × 10⁻⁸, 10⁻⁷, and 2.25 × 10⁻¹⁰ mutation per bacterium per generation. The relatively high mutation rate to ethambutol resistance and the low mutation rate to rifampin resistance were confirmed by analyzing the increase in the proportion of mutants with time in a growing population of the tubercle bacilli. The highest proportions of mutants to be expected in unselected populations of the tubercle bacilli were calculated from the results of fluctuation tests.     

ahpC, a gene involved in isoniazid resistance of the Mycobacterium tuberculosis complex

A gene conferring low-level isoniazid (INH) resistance on Mycobacterium smegmatis was isolated from a cosmid library of the genome of an INH-resistant Mycobacterium bovis strain. The gene had good homology with ahpC , the product of which is a subunit of alkyl hydroperoxide reductase, and also with a family of thiol-specific antioxidant enzymes. A mutation was found in the promoter upon comparison with the equivalent DNA sequence from the INH-sensitive parent strain. Promoter sequences from other INH-sensitive and INH-resistant M. bovis and Mycobacterium tuberculosis strains were sequenced and the mutation was found only in the INH-resistant strains. An INH-resistant M. tuberculosis strain also had an additional mutation in the promoter region. The wild-type promoter and promoters with one and two mutations were ligated into a reporter plasmid containing the lacZ gene. The presence of the first mutation resulted in a sixfold induction of β-galactosidase activity, and the presence of both mutations caused a 10-fold induction. Increased expression of AhpC may account for some of the INH resistance of strains of the M. tuberculosis complex.     

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA) in detecting isoniazid (INH) and rifampicin (RIF) resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results.     

Effect of inhA and katG on isoniazid resistance and virulence of Mycobacterium bovis

Isoniazid (INH) resistance of the Mycobacterium tuberculosis Complex (MtbC) is associated with both loss of catalase activity and mutation of the inhA gene. However, the relative contributions of these changes to resistance and to the loss of virulence for guinea-pigs is unknown. In this study, a virulent strain of Mycobacterium bovis, a member of the MtbC., was exposed to increasing concentrations of INH. Two INH-resistant strains were produced which had lost catalase activity. Strain WAg405, which had a higher resistance to INH, also had a mutation in the inhA gene. This demonstrated that loss of catalase activity and mutation of inhA had a cumulative effect on INH resistance. When a functional katG gene was integrated into the genome of WAg405 the INH resistance was greatly reduced. This indicated that most of the resistance had been caused by loss of catalase activity. While the parent INH-sensitive strain was virulent for guinea-pigs, the INH-resistant strains were significantly less virulent. Integration of a functional katG gene into the most resistant strain restored full virulence. This clearly established that katG is a virulence factor for M. bovis and that mutation of the inhA gene has no effect on virulence.     

Quantifying the Diversification of Hepatitis C Virus (HCV) during Primary Infection: Estimates of the In Vivo Mutation Rate

Hepatitis C virus (HCV) is present in the host with multiple variants generated by its error prone RNA-dependent RNA polymerase. Little is known about the initial viral diversification and the viral life cycle processes that influence diversity. We studied the diversification of HCV during acute infection in 17 plasma donors, with frequent sampling early in infection. To analyze these data, we developed a new stochastic model of the HCV life cycle. We found that the accumulation of mutations is surprisingly slow: at 30 days, the viral population on average is still 46% identical to its transmitted viral genome. Fitting the model to the sequence data, we estimate the median in vivo viral mutation rate is 2.5×10⁻⁵ mutations per nucleotide per genome replication (range 1.6–6.2×10⁻⁵), about 5-fold lower than previous estimates. To confirm these results we analyzed the frequency of stop codons (N = 10) among all possible non-sense mutation targets (M = 898,335), and found a mutation rate of 2.8–3.2×10⁻⁵, consistent with the estimate from the dynamical model. The slow accumulation of mutations is consistent with slow turnover of infected cells and replication complexes within infected cells. This slow turnover is also inferred from the viral load kinetics. Our estimated mutation rate, which is similar to that of other RNA viruses (e.g., HIV and influenza), is also compatible with the accumulation of substitutions seen in HCV at the population level. Our model identifies the relevant processes (long-lived cells and slow turnover of replication complexes) and parameters involved in determining the rate of HCV diversification.     

Genome-wide screen identifies rs646776 near sortilin as a regulator of progranulin levels in human plasma

Recent studies suggest progranulin (GRN) is a neurotrophic factor. Loss-of-function mutations in the progranulin gene (GRN) cause frontotemporal lobar degeneration (FTLD), a progressive neurodegenerative disease affecting ∼10% of early-onset dementia patients. Using an enzyme-linked immunosorbent assay, we previously showed that GRN is detectable in human plasma and can be used to predict GRN mutation status. This study also showed a wide range in plasma GRN levels in non-GRN mutation carriers, including controls. We have now performed a genome-wide association study of 313,504 single-nucleotide polymorphisms (SNPs) in 533 control samples and identified on chromosome 1p13.3 two SNPs with genome-wide significant association with plasma GRN levels (top SNP rs646776; p = 1.7 × 10⁻³⁰). The association of rs646776 with plasma GRN levels was replicated in two independent series of 508 controls (p = 1.9 × 10⁻¹⁹) and 197 FTLD patients (p = 6.4 × 10⁻¹²). Overall, each copy of the minor C allele decreased GRN levels by ∼15%. SNP rs646776 is located near sortilin (SORT1), and the minor C allele of rs646776 was previously associated with increased SORT1 mRNA levels. Supporting these findings, overexpression of SORT1 in cultured HeLa cells dramatically reduced GRN levels in the conditioned media, whereas knockdown of SORT1 increased extracellular GRN levels. In summary, we identified significant association of a locus on chromosome 1p13.3 with plasma GRN levels through an unbiased genome-wide screening approach and implicated SORT1 as an important regulator of GRN levels. This finding opens avenues for future research into GRN biology and the pathophysiology of neurodegenerative diseases.     

Mode of Binding of the Tuberculosis Prodrug Isoniazid to Heme Peroxidases: BINDING STUDIES AND CRYSTAL STRUCTURE OF BOVINE LACTOPEROXIDASE WITH ISONIAZID AT 2.7 ? RESOLUTION*

Isoniazid (INH) is an anti-tuberculosis prodrug that is activated by mammalian lactoperoxidase and Mycobacterium tuberculosis catalase peroxidase (MtCP). We report here binding studies, an enzyme assay involving INH, and the crystal structure of the complex of bovine lactoperoxidase (LPO) with INH to illuminate binding properties and INH activation as well as the mode of diffusion and interactions together with a detailed structural and functional comparison with MtCP. The structure determination shows that isoniazid binds to LPO at the substrate binding site on the distal heme side. The substrate binding site is connected to the protein surface through a long hydrophobic channel. The acyl hydrazide moiety of isoniazid interacts with Phe⁴²² O, Gln⁴²³ O^ϵ1, and Phe²⁵⁴ O. In this arrangement, pyridinyl nitrogen forms a hydrogen bond with a water molecule, W-1, which in turn forms three hydrogen bonds with Fe³⁺, His¹⁰⁹ N^ϵ2, and Gln¹⁰⁵ N^ϵ2. The remaining two sides of isoniazid form hydrophobic interactions with the atoms of heme pyrrole ring A, C^β and C^γ atoms of Glu²⁵⁸, and C^γ and C^δ atoms of Arg²⁵⁵. The binding studies indicate that INH binds to LPO with a value of 0.9 × 10⁻⁶ m for the dissociation constant. The nitro blue tetrazolium reduction assay shows that INH is activated by the reaction of LPO-H₂O₂ with INH. This suggests that LPO can be used for INH activation. It also indicates that the conversion of INH into isonicotinoyl radical by LPO may be the cause of INH toxicity.     

Antibiotic Resistance in Mycobacterium tuberculosis: PEROXIDASE INTERMEDIATE BYPASS CAUSES POOR ISONIAZID ACTIVATION BY THE S315G MUTANT OF M. TUBERCULOSIS CATALASE-PEROXIDASE (KatG)*

KatG (catalase-peroxidase) in Mycobacterium tuberculosis is responsible for activation of isoniazid (INH), a pro-drug used to treat tuberculosis infections. Resistance to INH is a global health problem most often associated with mutations in the katG gene. The origin of INH resistance caused by the KatG[S315G] mutant enzyme is examined here. Overexpressed KatG[S315G] was characterized by optical, EPR, and resonance Raman spectroscopy and by studies of the INH activation mechanism in vitro. Catalase activity and peroxidase activity with artificial substrates were moderately reduced (50 and 35%, respectively), whereas the rates of formation of oxyferryl heme:porphyrin π-cation radical and the decay of heme intermediates were ∼2-fold faster in KatG[S315G] compared with WT enzyme. The INH binding affinity for the resting enzyme was unchanged, whereas INH activation, measured by the rate of formation of an acyl-nicotinamide adenine dinucleotide adduct considered to be a bactericidal molecule, was reduced by 30% compared with WT KatG. INH resistance is suggested to arise from a redirection of catalytic intermediates into nonproductive reactions that interfere with oxidation of INH. In the resting mutant enzyme, a rapid evolution of 5-c heme to 6-c species occurred in contrast with the behavior of WT KatG and KatG[S315T] and consistent with greater flexibility at the heme edge in the absence of the hydroxyl of residue 315. Insights into the effects of mutations at residue 315 on enzyme structure, peroxidation kinetics, and specific interactions with INH are presented.Tuberculosis infection kills nearly 2 million people a year and is the leading cause of death due to infectious diseases in adults and in AIDS patients (1). The infection is usually treatable, and isoniazid (isonicotinic acid hydrazide (INH))⁴ has been a first line antibiotic against Mycobacterium tuberculosis since 1952 (2). The management of the disease is complicated by the fact that bacterial strains have been steadily acquiring and accumulating mutations that confer resistance to INH and other drugs (3–6). Recently, the appearance of multidrug-resistant tuberculosis, resistant to at least two first line antibiotics, and extensively drug-resistant bacteria (defined as multidrug-resistant tuberculosis plus resistance to at least one fluoroquinolone and at least one of the injectable second line drugs) has made the disease virtually incurable in a growing number of cases (7, 8). Despite the widespread emergence of antibiotic-resistant strains, the molecular mechanisms by which enzyme targets or pro-drug activating enzymes confer resistance are poorly understood.The pro-drug INH requires activation by M. tuberculosis catalase-peroxidase KatG, a heme enzyme classified in the Class I superfamily of fungal, plant, and bacterial peroxidases (9). KatG is important for the virulence of M. tuberculosis due to its role in oxidative stress management (10). This enzyme exhibits both high catalase activity and a broad spectrum peroxidase activity (9, 11) for which a physiologically relevant substrate has not been identified. In vitro, INH is oxidized by KatG (12–15) to an acylating species, most likely an acyl radical, that forms an adduct (IN-NAD) when it reacts with NAD⁺ (16). This modified cofactor then acts as a potent inhibitor of the M. tuberculosis enoyl-acyl carrier protein reductase, InhA, and interferes with cell wall biosynthesis (17, 18). The most common INH resistance mutations in M. tuberculosis clinical isolates occur in katG (19), although mutations in other genes, including inhA, and the promoter for this enzyme (mabA-inhA operon) may cause resistance (20–22). Dihydrofolate reductase has also been recently proposed as a target of isoniazid that can be inhibited by an IN-NADP adduct (23, 24). Issues remain to be resolved about INH action as well as resistance in a large set of clinical isolates.Replacements at residue Ser³¹⁵ are the most commonly encountered in the mutated katG gene of INH-resistant strains (19, 22, 25–28). Among these, S315T, which confers high level drug resistance (up to a 200-fold increase in minimum inhibitory concentration (MIC) that kills 50% of bacteria (29)) is the most frequent and is found in more than 50% of INH-resistant isolates of M. tuberculosis. In vitro, this mutant enzyme exhibits a very poor rate of peroxidation/activation of the antibiotic, although the enzyme has close to normal catalase activity and peroxidase activity with substrates other than INH (30–32). According to the crystal structure of KatG[S315T] (33), the replacement of serine by threonine leads to a structurally modified substrate access channel. This channel leads from the surface of the enzyme to the heme edge at the propionate of pyrrole IV. Residues Asp¹³⁷ and Ser³¹⁵ delimit the narrowest region of the channel, which is reduced in width from 6 to 4.7 Å. The methyl group of threonine effectively restricts accessibility to the heme pocket and apparently interferes with specific interactions required for binding and activation of the drug. Although a binding site for INH in KatG is not specifically defined by x-ray crystallography at this time, a recently reported CCP-INH structure (yeast CCP is a homologous Class I peroxidase) presents what should be an excellent model of drug binding in KatG (34). Hydrogen bonds between the backbone carbonyl of Ser¹⁸⁵ (Ser³¹⁵ in M. tuberculosis KatG), a water molecule, and the pyridine nitrogen of the drug are found in the CCP-INH complex. Thus, it is reasonable that mutations at residue 315 in KatG have an impact on drug binding and activation but little impact on catalase or peroxidase activity with substrates that may not require the same specific interactions as high affinity INH binding.Beyond these studies, there is a substantial gap in the knowledge of the relationship between INH resistance due to the numerous other mutations in the katG gene and the lost drug activation function of the mutant enzymes. The main goal of the present study was to examine KatG[S315G] in vitro. We report the generation, overexpression, purification, and characterization of this enzyme found in clinical isolates of M. tuberculosis having low level INH resistance with MIC values up to 40-fold higher than WT strains (8 μg/μl versus 0.05 μg/μl) (22, 25). An interesting aspect of the problem is that in KatG[S315T], a steric influence on INH binding strongly interferes with activation, whereas resistance is still present with the glycine replacement of serine 315, which would not be assumed to interfere with substrate access or binding at the same locus.The application of optical stopped-flow spectrophotometry, isothermal titration calorimetry (ITC), optical titration, EPR spectroscopy, and rapid freeze-quench EPR (RFQ-EPR) allowed us to probe the functional and structural consequences of the mutation on INH activation. Our results strongly suggest that resistance is due to catalytic changes rather than major changes in specific interactions between the enzyme and INH. Importantly, the results demonstrate the validity of an in vitro INH activation approach used here, since we find a correlation between our observations and the in vivo behavior of INH-resistant M. tuberculosis strains for both KatG[S315T] and KatG[S315G].     

Comparative analysis of TB-Biochip, Xpert MTB/RIF, and GenoType MTBDRplus test systems for rapid determination of mutations responsible for drug resistance of M. tuberculosis complex (in sputum from patients in Moscow region)

We studied the frequency of occurrence and combinations of mutations in rpoB, katG, inhA, and oxyR-ahpC genes of Mycobacterium tuberculosis (MTB) DNA isolated from patients of Moscow region. In isoniazid monoresistant MTB isolates, Ser315Thr mutation in the katG gene prevails (15.8%), whereas the most frequent mutations in multidrug-resistant MTB isolates were Ser531Leu in the rpoB gene, Ser315Thr in the katG gene (26.3%), and their combination with C(-15)T in the inhA gene (5.3%). The efficiency of TB-Biochip (OOO Biochip-IMB Russia), Xpert MTB/RIF (Cepheid, United States), and GenoType MTBDRplus (Hain Lifescience, Germany) test systems was analyzed and compared with the efficiency of luminescent microscopy and phenotypic drug-susceptibility testing in BACTEC? MGIT? 960 automated system (Becton, Dickinson and Company, United States). Using Xpert MTB/RIF, TB-Biochip, and GenoType MTBDRplus systems, MTB DNA was detected in sputum from patients in 92, 78, and 49% of all culturepositive cases, respectively. Standard cultural data match the test results of the susceptibility of MTB for Xpert MTB/RIF (rifampicin resistance) and for TB-Biochip and GenoType MTBDRplus (resistance to rifampicin and isoniazid) by 100, 97, and 100%, respectively. Thus, Xpert MTB/RIF system is the most efficient in primary MTB DNA detection, and TB-Biochip is the only one sensitive enough for both MTB DNA detection and determination of MTB multidrug resistance in sputum. Multidrug resistance is considered as resistance to both rifampicin and isoniazid.     

Comparison of Xpert MTB/RIF Assay and GenoType MTBDRplus DNA Probes for Detection of Mutations Associated with Rifampicin Resistance in Mycobacterium tuberculosis

BackgroundGeneXpert MTB/RIF (Xpert) and Genotype MTBDRplus (DRplus) are two World Health Organization (WHO) endorsed probe based molecular drug susceptibility testing (DST) methods for rapid diagnosis of drug resistant tuberculosis. Both methods target the same 81 bp Rifampicin Resistance Determining Region (RRDR) of bacterial RNA polymerase β subunit (rpoB) for detection of Rifampicin (RIF) resistance associated mutations using DNA probes. So there is a correspondence of the probes of each other and expected similarity of probe binding.MethodsWe analyzed 92 sputum specimens by Xpert, DRplus and LJ proportion method (LJ-DST). We compared molecular DSTs with gold standard LJ-DST. We wanted to see the agreement level of two molecular methods for detection of RIF resistance associated mutations. The 81bp RRDR region of rpoB gene of discrepant cases between the two molecular methods was sequenced by Sanger sequencing.ResultsThe agreement of Xpert and DRplus with LJ-DST for detection of RIF susceptibility was found to be 93.5% and 92.4%, respectively. We also found 92.4% overall agreement of two molecular methods for the detection of RIF susceptibility. A total of 84 out of 92 samples (91.3%) had agreement on the molecular locus of RRDR mutation by DRplus and Xpert. Sanger sequencing of 81bp RRDR revealed that Xpert probes detected seven of eight discrepant cases correctly and DRplus was erroneous in all the eight cases.ConclusionAlthough the overall concordance with LJ-DST was similar for both Xpert and DRplus assay, Xpert demonstrated more accuracy in the detection of RIF susceptibility for discrepant isolates compared with DRplus. This observation would be helpful for the improvement of probe based detection of drug resistance associated mutations especially rpoB mutation in M. tuberculosis.     

Estimation of the Spontaneous Mutation Rate per Nucleotide Site in a Drosophila melanogaster Full-Sib Family

We employed deep genome sequencing of two parents and 12 of their offspring to estimate the mutation rate per site per generation in a full-sib family of Drosophila melanogaster recently sampled from a natural population. Sites that were homozygous for the same allele in the parents and heterozygous in one or more offspring were categorized as candidate mutations and subjected to detailed analysis. In 1.23 × 10⁹ callable sites from 12 individuals, we confirmed six single nucleotide mutations. We estimated the false negative rate in the experiment by generating synthetic mutations using the empirical distributions of numbers of nonreference bases at heterozygous sites in the offspring. The proportion of synthetic mutations at callable sites that we failed to detect was <1%, implying that the false negative rate was extremely low. Our estimate of the point mutation rate is 2.8 × 10⁻⁹ (95% confidence interval = 1.0 × 10⁻⁹ − 6.1 × 10⁻⁹) per site per generation, which is at the low end of the range of previous estimates, and suggests an effective population size for the species of ∼1.4 × 10⁶. At one site, point mutations were present in two individuals, indicating that there had been a premeiotic mutation cluster, although surprisingly one individual had a G→A transition and the other a G→T transversion, possibly associated with error-prone mismatch repair. We also detected three short deletion mutations and no insertions, giving a deletion mutation rate of 1.2 × 10⁻⁹ (95% confidence interval = 0.7 × 10⁻⁹ − 11 × 10⁻⁹).     

Mycobacterium tuberculosis complex mycobacteria as amoeba-resistant organisms

Background

Most environmental non-tuberculous mycobacteria have been demonstrated to invade amoebal trophozoites and cysts, but such relationships are largely unknown for members of the Mycobacterium tuberculosis complex. An environmental source has been proposed for the animal Mycobacterium bovis and the human Mycobacterium canettii.

Methodology/Principal Findings

Using optic and electron microscopy and co-culture methods, we observed that 89±0.6% of M. canettii, 12.4±0.3% of M. tuberculosis, 11.7±2% of M. bovis and 11.2±0.5% of Mycobacterium avium control organisms were phagocytized by Acanthamoeba polyphaga, a ratio significantly higher for M. canettii (P = 0.03), correlating with the significantly larger size of M. canetti organisms (P = 0.035). The percentage of intraamoebal mycobacteria surviving into cytoplasmic vacuoles was 32±2% for M. canettii, 26±1% for M. tuberculosis, 28±2% for M. bovis and 36±2% for M. avium (P = 0.57). M. tuberculosis, M. bovis and M. avium mycobacteria were further entrapped within the double wall of <1% amoebal cysts, but no M. canettii organisms were observed in amoebal cysts. The number of intracystic mycobacteria was significantly (P = 10⁻⁶) higher for M. avium than for the M. tuberculosis complex, and sub-culturing intracystic mycobacteria yielded significantly more (P = 0.02) M. avium organisms (34×10⁴ CFU/mL) than M. tuberculosis (42×10¹ CFU/mL) and M. bovis (35×10¹ CFU/mL) in the presence of a washing fluid free of mycobacteria. Mycobacteria survived in the cysts for up to 18 days and cysts protected M. tuberculosis organisms against mycobactericidal 5 mg/mL streptomycin and 2.5% glutaraldehyde.

Conclusions/Significance

These data indicate that M. tuberculosis complex organisms are amoeba-resistant organisms, as previously demonstrated for non-tuberculous, environmental mycobacteria. Intercystic survival of tuberculous mycobacteria, except for M. canettii, protect them against biocides and could play a role in their life cycle.     

Whole Genome Sequencing of Mutation Accumulation Lines Reveals a Low Mutation Rate in the Social Amoeba Dictyostelium discoideum

Spontaneous mutations play a central role in evolution. Despite their importance, mutation rates are some of the most elusive parameters to measure in evolutionary biology. The combination of mutation accumulation (MA) experiments and whole-genome sequencing now makes it possible to estimate mutation rates by directly observing new mutations at the molecular level across the whole genome. We performed an MA experiment with the social amoeba Dictyostelium discoideum and sequenced the genomes of three randomly chosen lines using high-throughput sequencing to estimate the spontaneous mutation rate in this model organism. The mitochondrial mutation rate of 6.76×10⁻⁹, with a Poisson confidence interval of 4.1×10⁻⁹ − 9.5×10⁻⁹, per nucleotide per generation is slightly lower than estimates for other taxa. The mutation rate estimate for the nuclear DNA of 2.9×10⁻¹¹, with a Poisson confidence interval ranging from 7.4×10⁻¹³ to 1.6×10⁻¹⁰, is the lowest reported for any eukaryote. These results are consistent with low microsatellite mutation rates previously observed in D. discoideum and low levels of genetic variation observed in wild D. discoideum populations. In addition, D. discoideum has been shown to be quite resistant to DNA damage, which suggests an efficient DNA-repair mechanism that could be an adaptation to life in soil and frequent exposure to intracellular and extracellular mutagenic compounds. The social aspect of the life cycle of D. discoideum and a large portion of the genome under relaxed selection during vegetative growth could also select for a low mutation rate. This hypothesis is supported by a significantly lower mutation rate per cell division in multicellular eukaryotes compared with unicellular eukaryotes.