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 Impact of Isoniazid Resistance on the Treatment Outcomes of Smear Positive Re-Treatment Tuberculosis Patients in the State of Andhra Pradesh,India

Background

Multi drug resistant and rifampicin resistant TB patients in India are treated with the World Health Organization (WHO) recommended standardized treatment regimens but no guidelines are available for the management of isoniazid (INH) resistant TB patients. There have been concerns that the standard eight-month retreatment regimen being used in India (2H₃R₃Z₃E₃S₃/1H₃R₃Z₃E₃/5H₃R₃E₃; H-Isoniazid; R-Rifampicin; Z-Pyrazinamide; E-Ethambutol; S-Streptomycin) may be inadequate to treat INH resistant TB cases and leads to poor treatment outcomes. We aimed to assess if INH resistance is associated with unfavorable treatment outcomes (death, default, failure and transferred out) among a cohort of smear positive retreatment TB patients registered in three districts of Andhra Pradesh, India.

Methods

We conducted a retrospective record review of all smear positive retreatment TB patients without rifampicin resistance registered during April–December 2011.

Results

Of 1,947 TB patients, 1,127 (58%) were tested with LPA—50 (4%) were rifampicin resistant, 933 (84%) were sensitive to INH and rifampicin and 144 (12%) were INH resistant. Of 144 INH resistant cases, 64 (44%) had poor treatment outcomes (25 (17%) default, 22 (15%) death, 12 (8%) failure and 5 (3%) transfer out) as compared to 287 (31%) among INH sensitive cases [aRR 1.46; 95%CI (1.19–1.78)].

Conclusion

Our study confirms that INH resistance is independently associated with unfavorable treatment outcomes among smear positive retreatment TB patients, indicating that the current treatment regimen may be inadequate. These findings call for an urgent need for randomized controlled trials to discover the most effective treatment regimen for managing INH resistant TB.     

The use of macroarrays for the identification of MDR Mycobacterium tuberculosis

The emergence of Mycobacterium tuberculosis (Mtb), resistant to both isoniazid (INH) and rifampicin (RIF) (MDR-TB), is an increasing threat to tuberculosis control programs. Susceptibility testing of Mtb complex isolates by phenotypic methods requires a minimum of 14 days from a primary specimen. This can be reduced significantly if molecular analysis is used. Low density oligonucleotide arrays (macroarrays) have been used successfully for the detection of RIF resistance in Mtb. We describe the use of macroarray technology to identify Mtb complex isolates resistant to INH and/or RIF. The macroarray MDR-Mtb screen has been designed to detect mutations in the RIF resistance determining region (RRDR) of Mtb rpoB and loci in katG and mabA-inhA associated with INH resistance. A panel of Mtb isolates containing 38 different RRDR genotypes, 4 different genotypes within codon 315 of katG and 2 genotypes at mabA-inhA was used to validate the macroarray. The wild type (WT) genotype was correctly identified at all three loci. Of the 37 mutant rpoB genotypes, 36 were correctly detected; the single mutant not detected contained a 9 base insertion. All mutations within katG and mabA-inhA were correctly identified. We conclude that this low cost, rapid system can usefully detect the mutations associated with the vast majority of MDR-Mtb.     

Antimicrobial susceptibility determined by the E test, Löwenstein-Jensen proportion, and DNA sequencing methods among Mycobacterium tuberculosis isolates discrepancies, preliminary results

Mycobacterium tuberculosis strains resistant to streptomycin (SM), isoniazid (INH), and/or rifampin (RIF) as determined by the conventional L?wenstein-Jensen proportion method (LJPM) were compared with the E test, a minimum inhibitory concentration susceptibility method. Discrepant isolates were further evaluated by BACTEC and by DNA sequence analyses for mutations in genes most often associated with resistance to these drugs (rpsL, katG, inhA, and rpoB). Preliminary discordant E test results were seen in 75% of isolates resistant to SM and in 11% to INH. Discordance improved for these two drugs (63%) for SM and none for INH when isolates were re-tested but worsened for RIF (30%). Despite good agreement between phenotypic results and sequencing analyses, wild type profiles were detected on resistant strains mainly for SM and INH. It should be aware that susceptible isolates according to molecular methods might contain other mechanisms of resistance. Although reproducibility of the LJPM susceptibility method has been established, variable E test results for some M. tuberculosis isolates poses questions regarding its reproducibility particularly the impact of E test performance which may vary among laboratories despite adherence to recommended protocols. Further studies must be done to enlarge the evaluated samples and looked possible mutations outside of the hot spot sequenced gene among discrepant strains.     

Prevalence of mutations at codon 463 of katG gene in MDR and XDR clinical isolates of Mycobacterium tuberculosis in Belarus and application of the method in rapid diagnosis

Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. In respect to high GC content of Mycobacterium tuberculosis, nonsynonymous mutations are dominant in this group. In this study a collection of 145 M. tuberculosis isolates was used to evaluate the conferring mutations in nucleotide 1388 of katG gene (KatG463) in resistance to isoniazid. A PCR-RFLP method was applied in comparison with DNA sequencing and anti-mycobacterial susceptibility testing. From all studied patients, 98 (67.6%) were men, 47 (32.4%) were women, 3% were <15 and 9% were >65 years old; male to female ratio was 1:2.4. PCR result of katG for a 620-bp amplicon was successful for all purified M. tuberculosis isolates and there was no positive M. tuberculosis culture with PCR negative results (100% specificity). Subsequent PCR RFLP of the katG identified mutation at KatG463 in 33.3%, 57.8% and 59.2% of our clinically susceptible, multidrug resistant TB (MDR) and extensively drug resistant (XDR) isolates, respectively. Strains of H37Rv and Academic had no any mutations in this codon. M. bovis was used as a positive control for mutation in KatG463. Automated DNA sequencing of the katG amplicon from randomly selected INH-susceptible and resistant isolates verified 100% sequence accuracy of the point mutations detected by PCR-RFLP. We concluded that codon 463 was a polymorphic site that is associated to INH resistance (a missense or "quiet" mutation). RFLP results of katG amplicons were identical to those of sequence method. Our PCR-RFLP method has a potential application for rapid diagnosis of M. tuberculosis with a high specificity.     

Rifampicin Mono-Resistance in Mycobacterium tuberculosis in KwaZulu-Natal,South Africa: A Significant Phenomenon in a High Prevalence TB-HIV Region

Setting

The dual epidemics of HIV-TB including MDR-TB are major contributors to high morbidity and mortality rates in South Africa. Rifampicin (RIF) resistance is regarded as a proxy for MDR-TB. Currently available molecular assays have the advantage of rapidly detecting resistant strains of MTB, but the GeneXpert does not detect isoniazid (INH) resistance and the GenoTypeMTBDRplus(LPA) assay may underestimate resistance to INH. Increasing proportions of rifampicin mono-resistance resistance (RMR) have recently been reported from South Africa and other countries.

Objective

This laboratory based study was conducted at NHLS TB Laboratory, Durban, which is the reference laboratory for culture and susceptibility testing in KwaZulu-Natal. We retrospectively determined, for the period 2007 to 2009, the proportion of RMR amongst Mycobacterium tuberculosis (MTB) isolates, that were tested for both RIF and INH, using the gold standard of culture based phenotypic drug susceptibility testing (DST). Gender and age were also analysed to identify possible risk factors for RMR.

Design

MTB culture positive sputum samples from 16,748 patients were analysed for susceptibility to RIF and INH during the period 2007 to 2009. RMR was defined as MTB resistant to RIF and susceptible to INH. For the purposes of this study, only the first specimen from each patient was included in the analysis.

Results

RMR was observed throughout the study period. The proportion of RMR varied from a low of 7.3% to a high of 10.0% [overall 8.8%]. Overall, males had a 42% increased odds of being RMR as compared to females. In comparison to the 50 plus age group, RMR was 37% more likely to occur in the 25–29 year age category.

Conclusion

We report higher proportions of RMR ranging from 7.3% to 10% [overall 8.8%] than previously reported in the literature. To avoid misclassification of RMR, detected by the GeneXpert, as MDR-TB, culture based phenotypic DST must be performed on a second specimen, as recommended by the SA NDOH TB guidelines as well as WHO. We suggest that two sputum samples should be obtained at the first visit. The second sputum sample should be stored at 4°C. The latter sample is then readily available for performing additional DST (phenotypic or genotypic) for 2nd lines drugs, resulting in a decreased waiting period for DST results to become available.     

Do We Need to Detect Isoniazid Resistance in Addition to Rifampicin Resistance in Diagnostic Tests for Tuberculosis?

Background

Multidrug-resistant tuberculosis (MDR-TB) is resistant to both rifampicin (RIF) and isoniazid (INH). Whereas many TB diagnostics detect RIF-resistance, few detect INH-monoresistance, which is common and may increase risk of acquired MDR-TB. Whether inclusion of INH-resistance in a first-line rapid test for TB would have an important impact on MDR-TB rates remains uncertain.

Methods

We developed a transmission model to evaluate three tests in a population similar to that of India: a rapid molecular test for TB, the same test plus RIF-resistance detection (“TB+RIF”), and detection of RIF and INH-resistance (“TB+RIF/INH”). Our primary outcome was the prevalence of INH-resistant and MDR-TB at ten years.

Results

Compared to the TB test alone and assuming treatment of all diagnosed MDR cases, the TB+RIF test reduced the prevalence of MDR-TB among all TB cases from 5.5% to 3.8% (30.6% reduction, 95% uncertainty range, UR: 17–54%). Despite using liberal assumptions about the impact of INH-monoresistance on treatment outcomes and MDR-TB acquisition, expansion from TB+RIF to TB+RIF/INH lowered this prevalence only from 3.8% to 3.6% further (4% reduction, 95% UR: 3–7%) and INH-monoresistant TB from 15.8% to 15.1% (4% reduction, 95% UR: (-8)-19%).

Conclusion

When added to a rapid test for TB plus RIF-resistance, detection of INH-resistance has minimal impact on transmission of TB, MDR-TB, and INH-monoresistant TB.     

Impact of reduced vancomycin susceptibility on the therapeutic outcome of MRSA bloodstream infections

Background

Resistance to anti-tuberculosis drugs is a serious public health problem. Multi-drug resistant tuberculosis (MDR-TB), defined as resistance to at least rifampicin and isoniazid, has been reported in all regions of the world. Current phenotypic methods of assessing drug susceptibility of M. tuberculosis are slow. Rapid molecular methods to detect resistance to rifampicin have been developed but they are not affordable in some high prevalence countries such as those in sub Saharan Africa. A simple multi-well plate assay using mycobacteriophage D29 has been developed to test M. tuberculosis isolates for resistance to rifampicin. The purpose of this study was to investigate the performance of this technology in Kampala, Uganda.

Methods

In a blinded study 149 M. tuberculosis isolates were tested for resistance to rifampicin by the phage assay and results compared to those from routine phenotypic testing in BACTEC 460. Three concentrations of drug were used 2, 4 and 10 μg/ml. Isolates found resistant by either assay were subjected to sequence analysis of a 81 bp fragment of the rpoB gene to identify mutations predictive of resistance. Four isolates with discrepant phage and BACTEC results were tested in a second phenotypic assay to determine minimal inhibitory concentrations.

Results

Initial analysis suggested a sensitivity and specificity of 100% and 96.5% respectively for the phage assay used at 4 and 10 μg/ml when compared to the BACTEC 460. However, further analysis revealed 4 false negative results from the BACTEC 460 and the phage assay proved the more sensitive and specific of the two tests. Of the 39 isolates found resistant by the phage assay 38 (97.4%) were found to have mutations predictive of resistance in the 81 bp region of the rpoB gene. When used at 2 μg/ml false resistant results were observed from the phage assay. The cost of reagents for testing each isolate was estimated to be 1.3US$ when testing a batch of 20 isolates on a single 96 well plate. Results were obtained in 48 hours.

Conclusion

The phage assay can be used for screening of isolates for resistance to rifampicin, with high sensitivity and specificity in Uganda. The test may be useful in poorly resourced laboratories as a rapid screen to differentiate between rifampicin susceptible and potential MDR-TB cases.     

High Isoniazid Resistance Rates in Rifampicin Susceptible Mycobacterium tuberculosis Pulmonary Isolates from Pakistan

Background

Rapid new diagnostic methods (including Xpert MTB/RIF assay) use rifampicin resistance as a surrogate marker for multidrug resistant tuberculosis. Patients infected with rifampicin susceptible strains are prescribed first line anti-tuberculosis therapy. The roll out of such methods raises a concern that strains with resistance to other first line anti-tuberculosis drugs including isoniazid will be missed and inappropriate treatment given. To evaluate implications of using such methods review of resistance data from high burden settings such as ours is essential.

Objective

To determine resistance to first line anti-tuberculosis drugs amongst rifampicin susceptible pulmonary Mycobacterium tuberculosis (MTB) isolates from Pakistan.

Materials and Methods

Data of pulmonary Mycobacterium tuberculosis strains isolated in Aga Khan University Hospital (AKUH) laboratory (2009–2011) was retrospectively analyzed. Antimicrobial susceptibility profile of rifampicin susceptible isolates was evaluated for resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin.

Results

Pulmonary specimens submitted to AKUH from 2009 to 2011 yielded 7738 strains of Mycobacterium tuberculosis. These included 54% (n 4183) rifampicin susceptible and 46% (n: 3555) rifampicin resistant strains. Analysis of rifampicin susceptible strains showed resistance to at least one of the first line drugs in 27% (n:1133) of isolates. Overall isoniazid resistance was 15.5% (n: 649), with an isoniazid mono-resistance rate of 4% (n: 174). Combined resistance to isoniazid, pyrazinamide, and ethambutol was noted in 1% (n: 40), while resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin was observed in 1.7% (n: 70) of strains.

Conclusions

Our data suggests that techniques (including Xpert MTB/RIF assay) relying on rifampicin susceptibility as an indicator for initiating first line therapy will not detect patients infected with MTB strains resistant to other first line drugs (including isoniazid). The roll out of these techniques must therefore be accompanied by strict monitoring ensuring early resistance detection to increase chances of improved patient outcomes.     

Characterization of rpoB mutations associated with rifampin resistance in Mycobacterium tuberculosis from eastern China

Aims:  The aim of this study was to investigate the features of rpoB gene mutations associated with Rifampin (RIF) resistance in Mycobacterium tuberculosis ( M. tuberculosis ) in eastern China.
Methods and Results:  The mutations of rpoB gene in 56 clinical isolates of M. tuberculosis resisted to one to four first-line drugs (rifampin, isonicotinyl hydrazide, ethambutol and streptomycin) were analysed by polymerase chain reaction single strand conformation polymorphism analysis (PCR-SSCP) and DNA sequencing. The results of PCR-SSCP showed 52 isolates were positive (existing rpoB mutation) including 47 isolates resisted to RIF. Subsequent results of DNA sequencing showed that 54 isolates had rpoB gene mutation including 49 isolates resisted to RIF. The most frequently mutated sites were at codons 526 (73·2%), 513 (10·7%) and 531 (3·5%).
Conclusions:  The rpoB codon 526 was the most frequently mutated site of RIF-resistant M. tuberculosis strains in eastern China and its frequency is significantly higher ( P  < 0·0001) compared with that in other areas of China and in other geographic regions worldwide.
Significance and Impact of the Study:  Our results reveal that geographic variation is responsible for rpoB mutations in M. tuberculosis and the resulting information will be helpful to improve a novel rapid molecular drug resistance screening approach for MDR TB.     

A newly identified 191A/C mutation in the Rv2629 gene that was significantly associated with rifampin resistance in Mycobacterium tuberculosis

In an effort to identify the new resistance factors in rifampin resistant (RIF (r)) Mycobacterium tuberculosis ( M. tb), comparative proteome analysis and gene mutation assays were used to identify the differentially expressed proteins and correlated gene mutations among clinical RIF (r) isolates lacking rpoB mutations, RIF sensitive (RIF (s)) isolates, and the laboratory H37Rv strain. MALDI-TOF-MS revealed nine differentially expressed protein spots. PCR sequencing results showed four genes were mutated. The newly identified 191A/C mutation, in the gene Rv2629, was carried by 111 out of 112 clinical RIF (r) isolates. However, this mutation was absent in H37Rv and RIF (s) isolates. The RIF (s) species Mycobacterium smegmatis displayed RIF resistance only after being transformed with the mutated M. tb Rv2629, while it was not restored by the wild type gene. These results indicate that the 191A/C mutation of the Rv2629 gene may be associated with RIF resistance.     

Evaluation of BACTEC MGIT 960 System for Testing Susceptibility of Mycobacterium tuberculosis to First-Line Drugs in China

Background

The purpose of this study was to evaluate the performance of the BACTEC MGIT 960 (M960) system compared with the proportion method (PM) on Löwenstein-Jensen (L-J) medium in a peripheral laboratory in China for the testing of Mycobacterium tuberculosis (MTB) susceptibility to streptomycin (SM), isoniazid (INH) rifampicin (RIF) and ethambutol (EMB) a combination known as SIRE.

Methods

The susceptibility of 205 clinical isolates of MTB to SM, INH, RIF and EMB was performed with the M960 system. The drugs were tested at the following concentrations: 1.0 µg/ml for SM, 0.1 µg/ml for INH, 1.0 µg/ml for RIF, and 5.0 µg/ml for EMB. The results were compared with those obtained by the L-J PM. The L-J PM at an arbiter site was used to resolve any discordant results.

Results

The overall consistency was 96.6% and concordance values were 95.6% for SM, 97.6% for INH, 98.0% for RIF and 95.1% for EMB. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the M960 system for PM (the standard method) was 95.6%, 97.3%, 96.2% and 96.9% respectively, and the sensitivity were 93.3% for SM, 96.9% for INH, 97.4% for RIF and 94.6% for EMB, the specificity were 96.9% for SM, 98.2% for INH, 98.4% for RIF and 95.5% for EMB, the PPV were 94.6% for SM, 97.9% for INH, 97.4% for RIF and 94.6% for EMB, the NPV were 96.2% for SM, 97.3% for INH, 98.4% for RIF and 95.5% for EMB. The turnaround time with the M960 system (median 8.0 days, ranged from 5 to 14 days) was significantly shorter than that with the PM (28 days or 42 days).

Conclusion

There was a substantial degree of agreement between the two methods. The M960 system was a reliable and rapid method for SIRE susceptibility testing of tuberculosis in China.     

Characterization of mutations causing rifampicin and isoniazid resistance of Mycobacterium tuberculosis in Syria

In order to characterize mutations causing rifampicin and isoniazid resistance of M. tuberculosis in Syria, 69 rifampicin resistant (Rif(r)) and 72 isoniazid resistant (Inh(r)) isolates were screened for point mutations in hot spots of the rpoB, katG and inhA genes by DNA sequencing and real time PCR. Of 69 Rif(r) isolates, 62 (90%) had mutations in the rifampin resistance determining region (RRDR) of the rpoB gene, with codons 531 (61%), 526 (13%), and 516 (8.7%) being the most commonly mutated. We found two new mutations (Asp516Thr and Ser531Gly) described for the first time in the rpoB-RRDR in association with rifampicin resistance. Only one mutation (Ile572Phe) was found outside the rpoB-RRDR. Of 72 Inh(r) strains, 30 (41.6%) had a mutation in katGcodon315 (with Ser315Thr being the predominant alteration), and 23 (32%) harbored the inhA(-15C-->T) mutation. While the general pattern of rpoB-RRDR and katG mutations reflected those found worldwide, the prevalence of the inhA(-15C-->T mutation was above the value found in most other countries, emphasizing the great importance of testing the inhA(-15C-->T) mutation for prediction of isoniazid resistance in Syria. Sensitivity of a rapid test using real time PCR and 3'-Minor groove binder (MGB) probes in detecting Rif(r) and Inh(r) isolates was 90% and 69.4%, respectively. This demonstrates that a small set of MGB-probes can be used in real time PCR in order to detect most mutations causing resistance to rifampicin and isoniazid.     

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 evaluation of the microplate nitrate reductase assay and the rezasurin microtitre assay for the rapid detection of multidrug resistant Mycobacterium tuberculosis clinical isolates

The microplate nitrate reductase assay (MNRA) and the rezasurin microtitre assay (REMA) were used for the susceptibility testing of 73 clinical isolates and the results were compared with those that were obtained using the Bactec 460 TB and Bactec MGIT 960 systems. The REMA and the MNRA were performed in 96-well plates. For the REMA, the concentrations of isoniazid (INH) and rifampicin (RIF) ranged from 1.0-0.01 μg/mL and 2.0-0.03 μg/mL, respectively. For the MNRA, the INH concentration was between 1.0-0.03 μg/mL and the RIF concentration was between 2.0-0.06 μg/mL. For the MNRA, the sensitivity, specificity, positive predictive value, negative predictive value and INH/RIF agreement were 100/95.6, 97.6/100, 96.8/100, 100/98 and 98.6/98.6, respectively, and for the REMA, they were 100/91.3, 90.4/100, 88.5/100, 100/96.1 and 94.5/97.2, respectively. Our data suggest that these two rapid, low-cost methods may be inexpensive, alternative assays for the rapid detection of multidrug resistant tuberculosis in low-income countries.     

EnzyBase: a novel database for enzybiotic studies

Background

Staphylococcus aureus (S. aureus) is a major nosocomial pathogen that causes a variety of infections and toxicoses. In recent years, the percentage of rifampicin-resistant S. aureus has increased rapidly in China. The aims of this study were to analyze 1) the level of rifampicin resistance in S. aureus and its correlation with mutations in the rpoB gene, and 2) the molecular characterization of rifampicin-resistant S. aureus isolates.

Results

88 rifampicin-resistant S. aureus isolates were collected for this study. Of the 88 isolates, 83 (94.3%) were high-level rifampicin resistant (MIC??8 mg/L) while the remaining 5 isolates (5.7%) had a low-level resistance to rifampicin (MIC, 2 to 4 mg/L). Four amino acid substitutions were found in the 88 isolates, which were 481His/Asn (95.5%), 466Leu/Ser (87.5%), 477Ala/Asp (6.8%) and 486Ser/Leu (4.5%) respectively. All mutations were found to be present in cluster I of the rpoB gene. The low-level resistant isolates were found to have only one mutation, while the high-level resistant isolates had at least two or more mutations. The most common multiple mutations were 481His/Asn+466Leu/Ser(92.8%,77/83). The other multiple mutations found were 481His/Asn+477Ala/Asp (6.0%,5/83), and 481His/Asn+466Leu/Ser+477Ala/Asp (1.2%,1/83). Out of 28 high-level rifampicin-resistant S. aureus isolates, three molecular types were found, namely, ST239-MRSA-III-spa t030 (25/28, 89.3%), ST239-MRSA-III-spa t021 (2/28, 7.1%), and ST239-MRSA-III-spa t045 (1/28, 3.6%).

Conclusions

Rifampicin resistance in S. aureus was closely associated with mutations in the rpoB gene. High-level rifampicin-resistant S. aureus is one of the most important features in Anhui Provincial Hospital, and high-level rifampicin resistance in S. aureus is associated with multiple mutations of rpoB gene. The prevalence of high-level rifampicin-resistant S. aureus in Anhui may be associated with the spread of the ST239-MRSA III-spa t030 clone.     

Molecular characterization of mutation associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis isolates

Nucleotide changes in catalase peroxidase (Kat G) gene and gene encoding the beta subunit of RNA polymerase (rpo B), responsible for isoniazid and rifampicin drug resistance were determined in the clinical isolates of Mycobacterium tuberculosis by PCR-RFLP, Line probe assay and DNA sequencing. PCR-RFLP test was performed by HapII cleavage of an amplified fragment of Kat G gene to detect the transversion 315AGC-->ACC(Ser-->Thr) which is associated with INH drug resistance. The Line probe assay kit was evaluated to detect the mutation in 81bp RMP resistance determining region of rpo B gene associated with RMP drug resistance. These results were validated by DNA sequencing and drug susceptibility test. Kat G S 315 T mutation was found in 74.19% strains of M. tuberculosis from Delhi. This mutation was not found in any of the susceptible strains tested. The line probe assay kit and DNA sequencing identified 18 isolates as RMP resistant with specific mutation, while one of the RMP resistant strain was identified as RMP susceptible, with a concordance of 94.73% with the phenotypic drug susceptibility result. Majority (8 of 19, 42.1%) of resistant isolates involved base changes at codon 531 of rpo B gene. Both PCR-RFLP and Line probe assay test can be used in many of the clinical microbiology laboratories for early detection of isoniazid and rifampicin drug resistance in clinical isolates of M. tuberculosis.     

Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF) to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC), 523(GGG/GGT), 526(CAC/TAC), 531(TCG/TTG), 511(CTG/TTG), and 512(AGC/TCG). This study demonstrated the high specificity (93.8%) and sensitivity (95.2%) of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.     

Enhancing TB Case Detection: Experience in Offering Upfront Xpert MTB/RIF Testing to Pediatric Presumptive TB and DR TB Cases for Early Rapid Diagnosis of Drug Sensitive and Drug Resistant TB

Background

Diagnosis of pulmonary tuberculosis (PTB) in children is challenging due to difficulties in obtaining good quality sputum specimens as well as the paucibacillary nature of disease. Globally a large proportion of pediatric tuberculosis (TB) cases are diagnosed based only on clinical findings. Xpert MTB/RIF, a highly sensitive and specific rapid tool, offers a promising solution in addressing these challenges. This study presents the results from pediatric groups taking part in a large demonstration study wherein Xpert MTB/RIF testing replaced smear microscopy for all presumptive PTB cases in public health facilities across India.

Methods

The study covered a population of 8.8 million across 18 programmatic sub-district level tuberculosis units (TU), with one Xpert MTB/RIF platform established at each study TU. Pediatric presumptive PTB cases (both TB and Drug Resistant TB (DR-TB)) accessing any public health facilities in study area were prospectively enrolled and tested on Xpert MTB/RIF following a standardized diagnostic algorithm.

Results

4,600 pediatric presumptive pulmonary TB cases were enrolled. 590 (12.8%, CI 11.8–13.8) pediatric PTB were diagnosed. Overall 10.4% (CI 9.5–11.2) of presumptive PTB cases had positive results by Xpert MTB/RIF, compared with 4.8% (CI 4.2–5.4) who had smear-positive results. Upfront Xpert MTB/RIF testing of presumptive PTB and presumptive DR-TB cases resulted in diagnosis of 79 and 12 rifampicin resistance cases, respectively. Positive predictive value (PPV) for rifampicin resistance detection was high (98%, CI 90.1–99.9), with no statistically significant variation with respect to past history of treatment.

Conclusion

Upfront access to Xpert MTB/RIF testing in pediatric presumptive PTB cases was associated with a two-fold increase in bacteriologically-confirmed PTB, and increased detection of rifampicin-resistant TB cases under routine operational conditions across India. These results suggest that routine Xpert MTB/RIF testing is a promising solution to present-day challenges in the diagnosis of PTB in pediatric patients.