rpoB Mutations Research Articles

Frequency of multi-drug resistance and mutations in Mycobacterium tuberculosis isolates from Punjab state of India

Data regarding prevalence of multi-drug resistant tuberculosis (MDR-TB) and associated common mutations is scarce from Punjab region. The study was designed to determine rate of MDR-TB among presumptive MDR-TB from Punjab and mutation patterns using GenoType MTBDRplus assay. Total of 812 consecutive sputum samples were received from January 2012 to July 2013, from 14 districts of Punjab at the National Reference Laboratory at New Delhi for diagnosis of MDR-TB as hand holding activity. Presumptive MDR-TB patients were identified on basis of criterion B defined by the programme. Smear positive and negatives patients were found to be 636/798 (79.7%) and 162/ 798 (20.3%) respectively. Total of 606 GenoType MTBDRplus tests were conducted and mutations in rpoB, kat G and inhA genes analyzed. Total of 94/606 (15.5%), 43/606 (7.1%) and 40/606 (6.6%) were found to be RIF and INH resistant, mono-RIF resistant and 40/606 (6.6%) mono-INH resistant respectively. Commonest known mutation for RIF in rpoB gene and INH in kat G gene was S531L (80/ 137; 58.4%) and S315T1 (119/134; 88.8%) respectively. Mutations in inhA were found in 21/134 (15.7%) strains. Average turn-around time (TAT) for dispatch of result toPunjab was 4.6days. Prevalence of RIF resistance in Punjab was found to be 22.6%. Common mutations for RIF and INH were similar to that in other regions of country. GenoType MTBDRplus was found to be useful assay for rapid detection of MDR-TB, responsible for determining better management of MDR-TB patients under the programme.

Frequency of multi-drug resistance and mutations in Mycobacterium tuberculosis isolates from Punjab state of India

Data regarding prevalence of multi-drug resistant tuberculosis (MDR-TB) and associated common mutations is scarce from Punjab region. The study was designed to determine rate of MDR-TB among presumptive MDR-TB from Punjab and mutation patterns using GenoType MTBDRplus assay. Total of 812 consecutive sputum samples were received from January 2012 to July 2013, from 14 districts of Punjab at the National Reference Laboratory at New Delhi for diagnosis of MDR-TB as hand holding activity. Presumptive MDR-TB patients were identified on basis of criterion B defined by the programme. Smear positive and negatives patients were found to be 636/798 (79.7%) and 162/ 798 (20.3%) respectively. Total of 606 GenoType MTBDRplus tests were conducted and mutations in rpoB, kat G and inhA genes analyzed. Total of 94/606 (15.5%), 43/606 (7.1%) and 40/606 (6.6%) were found to be RIF and INH resistant, mono-RIF resistant and 40/606 (6.6%) mono-INH resistant respectively. Commonest known mutation for RIF in rpoB gene and INH in kat G gene was S531L (80/ 137; 58.4%) and S315T1 (119/134; 88.8%) respectively. Mutations in inhA were found in 21/134 (15.7%) strains. Average turn-around time (TAT) for dispatch of result toPunjab was 4.6 days. Prevalence of RIF resistance in Punjab was found to be 22.6%. Common mutations for RIF and INH were similar to that in other regions of country. GenoType MTBDRplus was found to be useful assay for rapid detection of MDR-TB, responsible for determining better management of MDR-TB patients under the programme.

Phenotypic and Genotypic Analysis of Multidrug-Resistant Mycobacterium tuberculosis Isolates from Sudanese Patients.

Background. Currently, mutations in rpoB, KatG, and rrs genes and inhA promoter were considered to be involved in conferring resistance to rifampicin, isoniazid, and streptomycin in Mycobacterium tuberculosis (MTB). Objective. The aims of this study were to detect the prevalence of first-line tuberculosis (TB) drug resistance among a group of previously treated and newly detected TB patients, to determine the association between prevalence of multidrug resistance (MDR) and demographic information (age and sex), to explain genes correlated with MDR Mycobacterium tuberculosis, and to characterize MTB via 16S ribosomal RNA (16S rRNA) analysis. Methods. A hundred MTB isolates from Sudanese pulmonary TB patients were included in the study. The proportional method of drug susceptibility test was carried out on Löwenstein-Jensen media. Multiplex PCR of rpoB and KatG genes and inhA promoter was conducted; then rrs genes were amplified by conventional PCR and were sequenced. The sequences of the PCR product were compared with known rrs gene sequences in the GenBank database by multiple sequence alignment tools. Result. The prevalence of MDR was 14.7% among old cases and 5.3% among newly diagnosed cases. Conclusion. Mutations in rrs could be considered as a diagnostic marker.

Molecular Genetic Determination of MDR–TB Isolates from West Bengal, India

Tuberculosis (TB) is one of the leading causes of death from an infectious disease worldwide. India remains to be the country carrying highest burden of the disease. One of the potent problems of present days, in this field, is the emergence of Multi-Drug-Resistant Tuberculosis (MDR-TB). MDR is defined as resistance to isoniazid (INH) and rifampin (RIF). The spread of MDR-TB is one of the biggest challenges to global public health system. Thus, detection of MDR-TB strains is critically important for containment of global TB epidemics. In this study, we are determining the MDR incidence rate in the West Bengal state of India, which is one of the most TB prone areas of the world. The RIF-resistant mutations of Mycobacterium tuberculosis (MTB) were shown to map in the rpoB locus and INH-resistant mutations of MTB were shown to map in inhA and katG loci. Therefore, in the present study, we have detected MDR-TB strains by mapping rpoB, katG & inhA mutations. Our Line Probe Assay (LPA) based results from a vast pool of MTB organisms (carrying 3653 bonafide sputum positive patient isolates) indicated the presence of 14.37% MDR isolates which matches in general with the previously reported results from various parts of the world (approx. 12-16%). Therefore, we conclude that our MDR rate is generally comparable to that of the other investigations performed in the rest of the world. We also found that the male patients are more likely to contract the MDR strains than the female patients (1.97:1.0). This data also conforms to the global statistics.

Prevalence of Gene Mutations profiles by GenoType MTBDRplus/sl to First Line Antituberculosis Drugs and Clinical Characteristics in Drug Resistant Tuberculosis Patients Referred to the National Institute of Respiratory Diseases in Mexico City

BackgroundDrug resistance tuberculosis, specially MDR and XDR are a big challenge for diagnosis and treatment. In Mexico the prevalence of MDR is between 3–5%, a number probably underestimated due to lack of diagnostic tests for susceptibility. The National Institute of Respiratory Diseases in Mexico City is the national referral center for MDR/XDR tuberculosis. In our country there is no data about the gene mutations involved in drug resistance to first line antituberculosis treatment nor the clinical characteristics that accompany these findings.Objective: Evaluate the prevalence of genotyping profiles according to a line probe assay (LPA) in patients with drug resistance tuberculosis and their associated clinical characteristicsMethodsRetrospective cohort from 2010 to 2014 of M. tuberculosis isolates with any type of resistance to first line antituberculosis drugs identified by MGIT SIRE and in which GenoType MTBDRplus/sl were performed, we evaluate prevalence of genotyping profiles according to the LPA within the isolates and gather data from those with complete medical records to asses clinical characteristics.ResultsIn 52 and 33 isolates phenotyping and genotyping MTBDRplus/sl respectively were performed, 41 resistant to Isoniazid INH with 75% genotypic concordance, 33 resistant to rifampicin RIF with 75.6% concordance, 14 to streptomycin SM with 23% concordance and 10 to ethambutol EMB with 100% concordance, 54% MDR tuberculosis. The genotyping profile for RIF was absence of probes rpoB Wild Type 8 (WT) 57.7%, WT 7 30.8% and presence of rpoB mutation 3 (MUT) 19.2%. For INH absence of InhA WT2 48.1% and InhA WT1 19.2%. For EMB absence of embB WT1 30.8% and for SM absence of rrs WT1 (19%). Absence of InhA WT1 was associated with female (P = 0.01) and DM2 (P = 0.032) patients, other clinical/biochemical characteristics and mortality was not different in patients with o without the genotypic profile for each drug. Cavitary disease by CT was more frequent in patients with WT probe absence in RIF and INH than those who did not have a LPA suggestive of resistance for this drugs.ConclusionWild Type probe absense is the frequent finding in our isolates according to LPA in RIF, INH, EMB and SM, intrisic host factors and clinical characteristics seem not to be related to a particular resistant gene profile.Disclosures All authors: No reported disclosures.

Novel rapid PCR for the detection of Ile491Phe rpoB mutation of Mycobacterium tuberculosis, a rifampicin-resistance-conferring mutation undetected by commercial assays

ObjectivesNeither the liquid medium-based Bactec MGIT, nor commercial molecular assays such as the Xpert MTB/RIF and the MTBDRplus V2.0 assays are capable of detecting up to 30% of rifampicin-resistant Mycobacterium tuberculosis strains in Swaziland because of the large proportion of the rpoB Ile491Phe mutations. In other countries, the frequency of this mutation is thought to be low. MethodsWe designed a real-time multiplex allele-specific PCR assay to identify the rpoB Ile491Phe mutation responsible for these undetected resistant M. tuberculosis strains. ResultsThe technique showed 100% similarity with rpoB sequencing on a panel of 78 strains from Swaziland. ConclusionsWe propose that the detection of the rpoB Ile491Phe rpoB mutation should complement commercial assays for the diagnosis of rifampicin-resistant M. tuberculosis in routine conditions, particularly in countries where this specific mutation is frequent. The technique proposed in this paper is adapted for most reference laboratories.

Correlation of rpoB Mutations with Minimal Inhibitory Concentration of Rifampin and Rifabutin in Mycobacterium tuberculosis in an HIV/AIDS Endemic Setting, South Africa.

Treatment of tuberculosis (TB) and HIV co-infections is often complicated by drug-to-drug interactions between anti-mycobacterial and anti-retroviral agents. Rifabutin (RFB) is an alternative to rifampin (RIF) for TB regimens and is recommended for HIV patients concurrently receiving protease inhibitors because of reduced induction of CYP3A4. This study sought to determine the proportion of RFB susceptible isolates among RIF-resistant strains in a high HIV prevalence setting in South Africa. In addition, the study explored the association between rpoB mutations and minimum inhibitory concentrations (MIC) of RIF and RFB. A total of 189 multidrug resistant (MDR) Mycobacterium tuberculosis isolates from the Centre for Tuberculosis repository were analyzed. The MICs were determined using a MYCOTB Sensititre plate method and the rpoB gene was sequenced. Of the 189 MDR isolates, 138 (73%) showed resistance to both RIF and RFB, while 51 (27%) isolates were resistant to RIF but retained susceptibility to RFB. The S531L was the most frequent rpoB point mutation in 105/189 (56%) isolates, followed by H526Y in 27/189 (14%) isolates. Resistance to both RIF and RFB was found predominantly in association with mutations S531L (91/105, 87%), H526Y (20/27, 74%), and H526D (15/19, 79%), while D516V (15/17, 88%), and L533P (3/4, 75%) were found in RIF-resistant, RFB-susceptible isolates. This study has shown that up to 27% of MDR-TB patients in South Africa may benefit from a treatment regimen that includes RFB.

Frequency of mutations in rifampicin and isoniazid resistant isolates of M. tuberculosis: an analysis from Central India.

The spread of drug-resistant tuberculosis has challenged tuberculosis control strategies globally. The present study aims to analyze the frequency of mutations in rpoB, katG and inhA genes in strains of M. tuberculosis complex (MTBC) circulating in Central India. It is anticipated that the findings may provide a starting point to understand the evolutionary success of drug-resistant strains of MTBC in this region. Line probe assay was carried out on 720 consecutive sputum samples of MDR suspects from June 2012 to May 2013. Mutation frequencies in the rpoB, katG and inhA genes were analyzed. Mutations were identified in 269 (37.6%) samples, as follows: 55 (7.6%) samples had mutations conferring resistance to only isoniazid, 84 (11.6%) had mutations conferring resistance to only rifampicin and 130 (18%) isolates had mutations conferring resistance to both isoniazid and rifampicin. The most frequent mutation in the rpoB gene was at codon S531L, seen in 141 (19.5%) isolates. The most frequent mutation in the katG gene was at codon S315T1, seen in 151 (20.9%) isolates; and in the inhA gene at codon C15T, seen in 21 (2.9%) isolates. Some unidentified mutations were also observed. The patterns and the frequency of the mutations identified in this study indicate the most frequent mutations at S531L codon in the rpoB gene, S315T1 codon in the katG gene and C15T codon in the promoter region of the inhA gene. Controlling the emergence and spread of MDR TB requires an understanding of the evolution of these mutations.

Global Effect of rpoB Mutation on Protein Expression in Enterococcus faecium

Background: Enterococcus faecium is an important nosocomial pathogen and has developed resistance to multiple antibiotics, such as vancomycin. Nitrofurantoin is active against E. faecium and E. faecalis, including vanA- and vanB-positive isolates. Objectives: To investigate the role of rpoB mutation in E. faecium. Methods: The growth rate and reactive oxygen species (ROS) levels were determined. The proteomic profiles of E. faecium B42 and its Rifr mutant B42-R7 were also compared. Complement experiments were performed to elucidate the role of nitroreductase in nitrofurantoin resistance. Results: The Rifr mutant (B42-R7, rpoB H489D) of E. faecium had a lower growth rate and higher ROS levels. Overexpression of nitroreductase in E. faecium B42-R7 was observed in proteomic analyses. Enhanced expression of nitroreductase in Escherichia coli increased the sensitivity to nitrofurantoin. Conclusions: The rpoB mutation in E. faecium not only altered the susceptibility to rifampin, but also affected global protein expression, including nitroreductase. Nitroreductase expression in E. faecium B42-R7 might play a role in nitrofurantoin resistance.

Leprosy Drug Resistance Surveillance in Colombia: The Experience of a Sentinel Country.

An active search for Mycobacterium leprae drug resistance was carried out, 243 multibacillary patients from endemic regions of Colombia were included from 2004 to 2013 in a surveillance program. This program was a World Health Organization initiative for drug resistance surveillance in leprosy, where Colombia is a sentinel country. M. leprae DNA from slit skin smear and/or skin biopsy samples was amplified and sequenced to identify mutations in the drug resistance determining region (DRDR) in rpoB, folP1, gyrA, and gyrB, the genes responsible for rifampicin, dapsone and ofloxacin drug-resistance, respectively. Three isolates exhibited mutations in the DRDR rpoB gene (Asp441Tyr, Ser456Leu, Ser458Met), two in the DRDR folP1 gene (Thr53Ala, Pro55Leu), and one isolate exhibited mutations in both DRDR rpoB (Ser456Met) and DRDR folP1 (Pro55Leu), suggesting multidrug resistance. One isolate had a double mutation in folP1 (Thr53Ala and Thr88Pro). Also, we detected mutations outside of DRDR that required in vivo evaluation of their association or not with drug resistance: rpoB Arg505Trp, folP1 Asp91His, Arg94Trp, and Thr88Pro, and gyrA Ala107Leu. Seventy percent of M. leprae mutations were related to drug resistance and were isolated from relapsed patients; the likelihood of relapse was significantly associated with the presence of confirmed resistance mutations (OR range 20.1–88.7, p < 0.05). Five of these relapsed patients received dapsone monotherapy as a primary treatment. In summary, the current study calls attention to M. leprae resistance in Colombia, especially the significant association between confirmed resistance mutations and relapse in leprosy patients. A high frequency of DRDR mutations for rifampicin was seen in a region where dapsone monotherapy was used extensively.

Whole Genome Sequence Analysis of a Large Isoniazid-Resistant Tuberculosis Outbreak in London: A Retrospective Observational Study.

BackgroundA large isoniazid-resistant tuberculosis outbreak centred on London, United Kingdom, has been ongoing since 1995. The aim of this study was to investigate the power and value of whole genome sequencing (WGS) to resolve the transmission network compared to current molecular strain typing approaches, including analysis of intra-host diversity within a specimen, across body sites, and over time, with identification of genetic factors underlying the epidemiological success of this cluster.Methods and FindingsWe sequenced 344 outbreak isolates from individual patients collected over 14 y (2 February 1998–22 June 2012). This demonstrated that 96 (27.9%) were indistinguishable, and only one differed from this major clone by more than five single nucleotide polymorphisms (SNPs). The maximum number of SNPs between any pair of isolates was nine SNPs, and the modal distance between isolates was two SNPs. WGS was able to reveal the direction of transmission of tuberculosis in 16 cases within the outbreak (4.7%), including within a multidrug-resistant cluster that carried a rare rpoB mutation associated with rifampicin resistance. Eleven longitudinal pairs of patient pulmonary isolates collected up to 48 mo apart differed from each other by between zero and four SNPs. Extrapulmonary dissemination resulted in acquisition of a SNP in two of five cases. WGS analysis of 27 individual colonies cultured from a single patient specimen revealed ten loci differed amongst them, with a maximum distance between any pair of six SNPs. A limitation of this study, as in previous studies, is that indels and SNPs in repetitive regions were not assessed due to the difficulty in reliably determining this variation.ConclusionsOur study suggests that (1) certain paradigms need to be revised, such as the 12 SNP distance as the gold standard upper threshold to identify plausible transmissions; (2) WGS technology is helpful to rule out the possibility of direct transmission when isolates are separated by a substantial number of SNPs; (3) the concept of a transmission chain or network may not be useful in institutional or household settings; (4) the practice of isolating single colonies prior to sequencing is likely to lead to an overestimation of the number of SNPs between cases resulting from direct transmission; and (5) despite appreciable genomic diversity within a host, transmission of tuberculosis rarely results in minority variants becoming dominant. Thus, whilst WGS provided some increased resolution over variable number tandem repeat (VNTR)-based clustering, it was insufficient for inferring transmission in the majority of cases.

Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects.

Resistance to the fluoroquinolone drug ciprofloxacin is commonly linked to mutations that alter the drug target or increase drug efflux via the major AcrAB-TolC transporter. Very little is known about other mutations that might also reduce susceptibility to ciprofloxacin. We discovered that an Escherichia coli strain experimentally evolved for resistance to ciprofloxacin had acquired a mutation in rpoB, the gene coding for the β-subunit of RNA polymerase. The aim of this work was to determine whether this mutation, and other mutations in rpoB, contribute to ciprofloxacin resistance and, if so, by which mechanism. Independent lineages of E. coli were evolved in the presence of ciprofloxacin and clones from endpoint cultures were screened for mutations in rpoB. Ciprofloxacin-selected rpoB mutations were identified and characterized in terms of effects on susceptibility and mode of action. Mutations in rpoB were selected at a high frequency in 3 out of 10 evolved lineages, in each case arising after the occurrence of mutations affecting topoisomerases and drug efflux. All ciprofloxacin-selected rpoB mutations had a high fitness cost in the absence of drug, but conferred a competitive advantage in the presence of ciprofloxacin. RNA sequencing and quantitative RT-PCR analysis showed that expression of mdtK, encoding a multidrug efflux transporter, was significantly increased by the ciprofloxacin-selected rpoB mutations. The susceptibility phenotype was shown to depend on the presence of an active mdtK and a mutant rpoB allele. These data identify mutations in RNA polymerase as novel contributors to the evolution of resistance to ciprofloxacin and show that the phenotype is mediated by increased MdtK-dependent drug efflux.

Using whole genome sequencing to identify resistance determinants and predict antimicrobial resistance phenotypes for year 2015 invasive pneumococcal disease isolates recovered in the United States

Our whole genome sequence (WGS) pipeline was assessed for accurate prediction of antimicrobial phenotypes. For 2316 invasive pneumococcal isolates recovered during 2015 we compared WGS pipeline data to broth dilution testing (BDT) for 18 antimicrobials. For 11 antimicrobials categorical discrepancies were assigned when WGS-predicted MICs and BDT MICs predicted different categorizations for susceptibility, intermediate resistance or resistance, ranging from 0.9% (tetracycline) to 2.9% (amoxicillin). For β-lactam antibiotics, the occurrence of at least four-fold differences in MIC ranged from 0.2% (meropenem) to 1.0% (penicillin), although phenotypic retesting resolved 25%–78% of these discrepancies. Non-susceptibility to penicillin, predicted by penicillin-binding protein types, was 2.7% (non-meningitis criteria) and 23.8% (meningitis criteria). Other common resistance determinants included mef (475 isolates), ermB (191 isolates), ermB + mef (48 isolates), tetM (261 isolates) and cat (51 isolates). Additional accessory resistance genes (tetS, tet32, aphA-3, sat4) were rarely detected (one to three isolates). Rare core genome mutations conferring erythromycin-resistance included a two-codon rplD insertion (rplD69-KG-70) and the 23S rRNA A2061G substitution (six isolates). Intermediate cotrimoxazole-resistance was associated with one or two codon insertions within folP (238 isolates) or the folA I100L substitution (38 isolates), whereas full cotrimoxazole-resistance was attributed to alterations in both genes (172 isolates). The two levofloxacin-resistant isolates contained parC and/or gyrA mutations. Of 11 remaining isolates with moderately elevated MICs to both ciprofloxacin and levofloxacin, seven contained parC or gyrA mutations. The two rifampin-resistant isolates contained rpoB mutations. WGS-based antimicrobial phenotype prediction was an informative alternative to BDT for invasive pneumococci.

ГЕНЕТИЧЕСКИЕ ОСОБЕННОСТИ ВОЗБУДИТЕЛЯ ТУБЕРКУЛЕЗА В УРАЛЬСКОМ ФЕДЕРАЛЬНОМ ОКРУГЕ РОССИИ

The article presents molecular genetic description of isolates of Mycobacterium tuberculosis of 178 new patients and 78 patients who had been previously treated obtained in 2009-2011 on the territory of Urals. PCR in real time and MIRU-VNTR typing of 15 loci detected prevalence of M. tuberculosis of Beijing genotype in the both groups of patients, however the part of Beijing isolates was significantly higher in the group of patients who had been previously treated compared to new patients: 80.8% versus 55.1% (p = 0.0002). IS6110-RFLP-typing showed that the majority of Beijing isolates belonged to B0 clone, clinically and epidemiologically significant in Russia. It was found out that M. tuberculosis Beijing carrying mutations rpoB Ser531→Leu and katG Ser315→Thr played the major role in MDR tuberculosis transmission in Urals. Non-Beijing group was represented by isolates of various spoligotypes and MIRU-VNTR types among which the representatives of the following globally common genetic groups prevailed: LAM (LAM9, T5_RUS), URAL (Н3), Haarlem (H1, X), Т (Т1).

Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus.

Staphylococcus aureus is capable of causing a remarkable spectrum of disease, ranging from mild skin eruptions to life-threatening infections. The survival and pathogenic potential of S. aureus depend partly on its ability to sense and respond to changes in its environment. Spx is a thiol/oxidative stress sensor that interacts with the C-terminal domain of the RNA polymerase RpoA subunit, leading to changes in gene expression that help sustain viability under various conditions. Using genetic and deep-sequencing methods, we show that spx is essential in S. aureus and that a previously reported Δspx strain harbored suppressor mutations that allowed it to grow without spx One of these mutations is a single missense mutation in rpoB (a P-to-L change at position 519 encoded by rpoB [rpoB-P519L]) that conferred high-level resistance to rifampin. This mutation alone was found to be sufficient to bypass the requirement for spx The generation of rifampin resistance libraries led to the discovery of an additional rpoB mutation, R484H, which supported strains with the spx disruption. Other rifampin resistance mutations either failed to support the Δspx mutant or were recovered at unexpectedly low frequencies in genetic transduction experiments. The amino acid residues encoded by rpoB-P519L and -R484H map in close spatial proximity and comprise a highly conserved region of RpoB. We also discovered that multicopy expression of either trxA (encoding thioredoxin) or trxB (encoding thioredoxin reductase) supports strains with the deletion of spx Our results reveal intriguing properties, especially of RNA polymerase, that compensate for the loss of an essential gene that is a key mediator of diverse processes in S. aureus, including redox and thiol homeostasis, antibiotic resistance, growth, and metabolism. The survival and pathogenicity of S. aureus depend on complex genetic programs. An objective for combating this insidious organism entails dissecting genetic regulatory circuits and discovering promising new targets for therapeutic intervention. In this study, we discovered that Spx, an RNA polymerase-interacting stress regulator implicated in many stress responses in S. aureus, including responses to oxidative and cell wall antibiotics, is essential. We describe two mechanisms that suppress the lethality of spx disruption. One mechanism highlights how only certain rifampin resistance-encoding alleles of RpoB confer new properties on RNA polymerase, with important mechanistic implications. We describe additional stress conditions where the loss of spx is deleterious, thereby highlighting Spx as a multifaceted regulator and attractive drug discovery target.

Rifampin Resistance Mutations Are Associated with Broad Chemical Remodeling of Mycobacterium tuberculosis

Global control of tuberculosis has become increasingly complicated with the emergence of multidrug-resistant strains of Mycobacterium tuberculosis First-line treatments are anchored by two antibiotics, rifampin and isoniazid. Most rifampin resistance occurs through the acquisition of missense mutations in the rifampin resistance-determining region, an 81-base pair region encoding the rifampin binding site on the β subunit of RNA polymerase (rpoB). Although these mutations confer a survival advantage in the presence of rifampin, they may alter the normal process of transcription, thereby imposing significant fitness costs. Because the downstream biochemical consequences of the rpoB mutations are unknown, we used an organism-wide screen to identify the number and types of lipids changed after rpoB mutation. A new mass spectrometry-based profiling platform systematically compared ∼10,000 cell wall lipids in a panel of rifampin-resistant mutants within two genetically distinct strains, CDC1551and W-Beijing. This unbiased lipidomic survey detected quantitative alterations (>2-fold, p < 0.05) in more than 100 lipids in each mutant. By focusing on molecular events that change among most mutants and in both genetic backgrounds, we found that rifampin resistance mutations lead to altered concentrations of mycobactin siderophores and acylated sulfoglycolipids. These findings validate a new organism-wide lipidomic analysis platform for drug-resistant mycobacteria and provide direct evidence for characteristic remodeling of cell wall lipids in rifampin-resistant strains of M. tuberculosis The specific links between rifampin resistance and named lipid factors provide diagnostic and therapeutic targets that may be exploited to address the problem of drug resistance.

Epistatic interactions between ancestral genotype and beneficial mutations shape evolvability in Pseudomonas aeruginosa.

The idea that interactions between mutations influence adaptation by driving populations to low and high fitness peaks on adaptive landscapes is deeply ingrained in evolutionary theory. Here, we investigate the impact of epistasis on evolvability by challenging populations of two Pseudomonas aeruginosa clones bearing different initial mutations (in rpoB conferring rifampicin resistance, and the type IV pili gene network) to adaptation to a medium containing l-serine as the sole carbon source. Despite being initially indistinguishable in fitness, populations founded by the two ancestral genotypes reached different fitness following 300 generations of evolution. Genome sequencing revealed that the difference could not be explained by acquiring mutations in different targets of selection; the majority of clones from both ancestors converged on one of the following two strategies: (1) acquiring mutations in either PA2449 (gcsR, an l-serine-metabolism RpoN enhancer binding protein) or (2) protease genes. Additionally, populations from both ancestors converged on loss-of-function mutations in the type IV pili gene network, either due to ancestral or acquired mutations. No compensatory or reversion mutations were observed in RNA polymerase (RNAP) genes, in spite of the large fitness costs typically associated with mutations in rpoB. Although current theory points to sign epistasis as the dominant constraint on evolvability, these results suggest that the role of magnitude epistasis in constraining evolvability may be underappreciated. The contribution of magnitude epistasis is likely to be greatest under the biologically relevant mutation supply rates that make back mutations probabilistically unlikely.

A rifampicin-resistant (rpoB) mutation in Pseudomonas protegens Pf-5 strain leads to improved antifungal activity and elevated production of secondary metabolites

Ribosome engineering has proven to be a practical method for increasing antibiotic production, and is extensively applied to strain improvement in antibiotic production and activation of silent genes in several prokaryotes. In this study, ribosome engineering was used to improve production of bioactive secondary metabolites produced by Pseudomonas protegens Pf-5. Rifampicin-resistant mutants that bear the H531N in the β-subunit of RNA polymerase showed improved antifungal activity and morphological changes. The production of several secondary metabolites in R55 mutant was significantly improved using high-performance liquid chromatography (HPLC) analysis. Two antibiotics with antifungal activity, 2, 4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt), which may contribute to the improved antifungal activity of the R55 mutant, were identified by mass spectrometer (MS) analysis.

Clinical Impact on Tuberculosis Treatment Outcomes of Discordance Between Molecular and Growth-Based Assays for Rifampin Resistance, California 2003-2013.

Background. Data from international settings suggest that isolates of Mycobacterium tuberculosis with rpoB mutations testing phenotypically susceptible to rifampin (RIF) may have clinical significance. We analyzed treatment outcomes of California patients with discordant molecular-phenotypic RIF results.Methods. We included tuberculosis (TB) patients, during 2003–2013, whose specimens tested RIF susceptible phenotypically but had a rpoB mutation determined by pyrosequencing. Demographic data were abstracted from the California TB registry. Phenotypic drug-susceptibility testing, medical history, treatment, and outcomes were abstracted from medical records.Results. Of 3330 isolates tested, 413 specimens had a rpoB mutation (12.4%). Of these, 16 (3.9%) had molecular-phenotypic discordant RIF results. Seven mutations were identified: 511Pro, 516Phe, 526Asn, 526Ser (AGC and TCC), 526Cys, and 533Pro. Fourteen (88%) had isoniazid (INH) resistance, 6 of whom were also phenotypically resistant to ethambutol (EMB) and/or pyrazinamide (PZA). Five patients (25%), 1 with 511Pro and 4 with 526Asn, relapsed or failed treatment. The initial regimen for 3 patients was RIF, PZA, and EMB; 1 patient received RIF, PZA, EMB, and a fluoroquinolone (FQN); and 1 patient received RIF, EMB, FQN, and some second-line medications. Upon retreatment with an expanded regimen, 3 (75%) patients completed treatment, 1 patient moved before treatment completion, and 1 patient continues on treatment. The remaining 11 patients had a successful outcome with 9 having received a FQN and/or a rifamycin.Conclusions. Rifampin molecular-phenotypic discordance was rare, and most isolates had INH resistance. Patients who did not receive an expanded regimen had poor outcomes. These mutations may have clinical importance, and expanded treatment regimens should be considered.

Rapid Detection of Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis Using Real-Time PCR.

BackgroundAccurate and rapid detection of drug-resistant Mycobacterium tuberculosis is fundamental for the successful treatment of tuberculosis (TB).ObjectivesThe aim of this study was to determine the frequency of common mutations leading to isoniazid (INH) and rifampicin (RMP) resistance.Patients and MethodsIn a cross-sectional study carried out in 2014, 90 patients with M. tuberculosis from five border provinces of Iran were selected. After a full clinical history and physical evaluation, real-time polymerase chain reaction (PCR) technique was performed for the detection of mutations in the patients’ katG and rpoB genes. The results were compared with results of a standard proportion method as well as a multiplex allele-specific PCR (MAS-PCR).ResultsA total of 23 mutations were found in isolates among which, codon katG 315, rpoB P1 (511 - 519 sequence) and rpoB P2 (524-533 sequence) were responsible for seven, nine and seven cases, respectively. The mean (standard deviation (SD)) of melting temperature (Tm) in katG 315 codon, rpoB P1 and P2 sequences in susceptible and mutant isolates was as follows: katG 85.4°C (0.18) and 87.54°C (0.62); rpoΒ P1 84.6°C (0.61) and 82.9°C (0.38); rpoΒ P2 83.4°C (0.18) and 85.3°C (0.19), respectively. In comparison to the standard proportion test, the sensitivity of real-time PCR in detecting INH- and RMP-resistant mutations was 75% and 83.3%, respectively. In comparison to the MAS-PCR test, 100% of katG 315 mutations and 80% of rpoB mutations were determined. Overall, 10% of the patients were diagnosed with a recurrence of TB. Age and previous history of TB treatment increased mutation odds in rpoB sequences (P = 0.046, P = 0.036, respectively).ConclusionsDetection of drug resistance associated with mutations through real-time PCR by melting analysis technique showed a high differentiating power. This technique had high concordance with the standard proportion test and MAS-PCR results.