Rifampin Resistance-determining Region Research Articles

High-throughput mutagenesis and screening approach for the identification of drug-resistant mutations in the rifampicin resistance-determining region of mycobacteria

Rifampicin is the most powerful first-line antibiotic for tuberculosis, which is caused by Mycobacterium tuberculosis. Although accumulating evidence from sequencing data of clinical M. tuberculosis isolates suggested that mutations in the rifampicin-resistance-determining region (RRDR) are strongly associated with rifampicin resistance, the comprehensive characterisation of RRDR polymorphisms that confer this resistance remains challenging. By incorporating I-SceI sites for I-SceI-based integrant removal and utilizing an L5 swap strategy, we efficiently replaced the integrated plasmid with alternative alleles, making mass allelic exchange feasible in mycobacteria. Using this method to establish a fitness-related gain-of function screen, we generated a mutant library that included all single-amino-acid mutations in the RRDR, and identified the important positions corresponding to some well-known rifampicin-resistance mutations (Q513, D516, S522, H525, R529, S531). We also detected a novel two-point mutation located in the RRDR confers a fitness advantage to M. smegmatis in the presence or absence of rifampicin. Our method provides a comprehensive insight into the growth phenotypes of RRDR mutants and should facilitate the development of anti-tuberculosis drugs.

Application of the real-time PCR Taqman allelic discrimination assay for the detection of Isoniazid and/or Rifampicin resistant Mycobacterium Tuberculosis from clinical samples

Background: Drug-resistant Tuberculosis (DR-TB) is challenging public health problem in countries with high tuberculosis ​​prevalence and limited resources. Developing and applying the most appropriate and effective methods for diagnosing DR-TB from clinical samples is necessary, allowing a more rapid detection method for large-scale screening. Methods: Applying real-time PCR Taqman allelic discrimination with a PCR Taqman probes panel to identifying the DR-TB associated mutations in rpoB and katG of Mycobacterium Tuberculosis from isolates and clinical samples. Results: Comparing results of the real-time PCR allelic and DNA sequencing results, the sensitivity and specificity for Isoniazid resistance detection by analysing katG were found 95%(75.1 - 99.8) and 100%, Rifampicin resistance determining region (RRDR) of rpoB were found 95.5(77.16 - 99.88) and 100%, respectively. The real-time PCR TaqMan allelic discrimination also showed the sensitivities 100% for both katG and rpoB, and the specificities were 93.55% (78.58 - 99.21) for the rpoB and 93.94% (79.77 - 99.26) for the katG from clinical samples. Conclusions: This study showed that the real-time PCR taqman allelic discrimination assay is useful for detection of TB and DR-TB because of an accurate and rapid diagnosis in the early stages. Key words: rug-resistant, Tuberculosis, clinical samples, real-time PCR taqman allelic discrimination assay, Mycobacterium tuberculosis.

Designing a Cas9/gRNA-assisted quantitative Real-Time PCR (CARP) assay for identification of point mutations leading to rifampicin resistance in the human pathogen Mycobacterium tuberculosis

A simple, rapid and low-cost diagnostic test, which can detect both the drug-sensitive and the drug-resistant tuberculosis (TB) cases is the need of the hour. Here, we developed a Cas9/gRNA-assisted quantitative Real-Time PCR (qRT-PCR) (CARP) assay to detect single nucleotide mutations causing drug resistance in the TB pathogen, Mycobacterium tuberculosis (Mtb). Guide RNAs (gRNAs) were designed against S531 and H526 positions in the rifampicin (RIF)-resistance-determining region (RRDR) of the Mtb rpoB gene that exhibit frequent mutations in the RR clinical isolates of Mtb. Conditions were optimised for in vitro Cas9 cleavage such that single nucleotide changes at these positions can be recognised by Cas9/gRNA complex with high sensitivity and 100% specificity. Further estimation of Cas9/gRNA-based cleavage of target DNA by qRT-PCR led to rapid detection of drug-resistant sequences. The newly designed CARP assay holds a great deal of promise in the diagnosis and prognosis of patients suffering from TB, in a cost-effective manner.

Human brucellosis in the southern West Bank, 2015–17: a seroprevalence and molecular characterisation study

BackgroundBrucellosis is a global zoonotic disease ranked by WHO as being among the top seven neglected zoonoses. After implementing a livestock vaccination strategy in 1998, the incidence rate of human brucellosis in Palestine dropped significantly. However, in 2015 the disease re-emerged with alarming incidence rates similar to those in the period before 1998. The aim of this study was to characterise the strains associated with this episode using genetic and genomic tools. MethodsAll patients were referred to the department of preventive medicine from several public or private clinics in the southern West Bank due to undulant fever. After a careful analysis of a patients’ history to evaluate exposure to Brucella, blood samples from patients suspected of brucellosis were collected. Samples positive for the Rose Bengal test with an antibody titre of 1:80 IU/mL or more by Brucella standard agglutination test, and not under antibiotic treatment, were further cultured using the BACTEC 9240 blood culture system. DNA was extracted from the positive culture samples and analysed using three biomarkers to validate the presence of the Brucella genus, establish the species, and identify the Brucella melitensis Rev 1 vaccine strain. Nine randomly chosen isolates were sequenced using targeted next generation sequencing technology to screen for the rpoB gene mutations. Multiple-locus variable number of tandem repeats analysis (MLVA) with 16 variable number of tandem repeats markers was used to genotype the isolates and BioNumerics version 7.6 software was used to analyse the data using the categorical coefficient and unweighted pair group method using the arithmetic averages algorithm. FindingsBlood samples were obtained from a total of 1324 patients during 2015–17. 704 (53·2%) of 1324 serum samples showed agglutination in the Rose Bengal test and 559 (79·4%) of the 704 were considered seropositive based on a standard agglutination test titre of 1:80 IU/mL or more. Only 130 (23·3%) of the 559 seropositive samples were from patients not under antibiotic treatment. Upon culturing, only 72 (12·9%) of the seropositive samples showed growth signals and were positive by PCR for the Brucella genus marker, and all isolates were B melitensis. None of the samples were associated with the B melitensis Rev 1 vaccine strain. The results of sequencing the rpoB gene by next generation sequencing showed four different single nucleotide variations that were not located within the rifampicin resistance-determining region. Using MLVA-16 analysis and the MLVA bank benchmark, the studied isolates were divided into 22 unique genotypes. A hierarchical clustering analysis for the 72 isolates and 18 B melitensis global isolates representing four major regions in the Middle East, Europe, Africa, and America, clustered the 73 isolates in the Middle East clade, providing a similarity cutoff value of more than 97% for genotype and more than 75% for clade. InterpretationThis study shows that the brucellosis episode of 2015–17 was due to B melitensis local isolates and was not associated with the Rev 1 vaccine stain. The results show that MLVA-16 is a useful tool for the public health surveillance of human brucellosis. The present study analysed samples from the Hebron governorate only. It is recommended to include more human and animal samples from different areas in Palestine to understand the molecular epidemiology of brucellosis in Palestine and to make more effective decisions regarding control and treatment programmes. FundingPalestine Polytechnic University.

Identification of mutations in rpoB, pncA, embB, and ubiA genes among drug-resistant Mycobacterium tuberculosis clinical isolates from Iran.

Mycobacterium tuberculosis resistant to effective first-line drugs (FLDs) has challenged national and global tuberculosis control programs. This study aimed to identify mutations in 4 genes related to rifampin, pyrazinamide, and ethambutol resistance among clinical isolates of M. tuberculosis from southwestern Iran. After drug susceptibility testing of 6620 M. tuberculosis clinical isolates by proportional method, a total of 24 FLD-resistant strains were included in the study. Fragments of rpoB, pncA, embB, and ubiA genes were amplified and sequenced to mine the mutations by pairwise alignment with the corresponding M. tuberculosis H37Rv genes. Phenotypic resistance to rifampin, isoniazid, and ethambutol was detected in 67, 54, and 33% (n = 16, 13, and 8) of the isolates, respectively. Of rifampin-resistant isolates, 31% (5/16) were mono-resistant, and 56% (9/16) were multidrug-resistant (MDR). In 100% of rifampin-resistant isolates, mutations were found in the rifampin resistance-determining region (RRDR) of the rpoB, with S450L substitution being the most common, especially in MDRs (77.8%, 7/9). Resistance-conferring mutations in pncA were present in 12.5% (3/24) of FLD-resistant isolates. The embB and ubiA mutations were found in 62.5 and 12.5% (5/8 and 1/8) of ethambutol-resistant isolates, respectively, of which the embB D354A was the most common substitution (37.5%, 3/8). Sixteen distinct mutations were identified, one of which was novel. The sequence analysis of the RRDR segment was the best way to detect rifampin resistance. The rpoB S450L substitution could be a helpful molecular marker to predict MDR. In other genes, no mutation was identified as a reliable marker.

Molecular Characterization of Rifampicin-Resistant Staphylococcus aureus Isolates from Retail Foods in China.

This study investigated the molecular characteristics of rifampin-resistant (RIF-R) Staphylococcus aureus isolates recovered from 4300 retail food samples covering most provincial capitals in China, from 2011 to 2016. Of the 1463 S. aureus enrolled, 149 isolates (142 MSSA and 7 MRSA) were identified as rifampicin-resistant, including 20 high-level (MICs ≥ 8 μg/mL) and 129 low-level (MICs between 2 and 4 μg/mL) rifampicin-resistant strains. Most of the RIF-R S. aureus isolates were resistant to more than three antibiotics. The mutations in the rifampicin resistance-determining region of the rpoB gene were studied in all RIF-R strains. All of the strains presented the mutational change 481 His/Asn and five isolates presented an additional mutation, including 477 Asp/Tyr, 527 Ile/Met, and 466 Leu/Ser, respectively. Thirteen STs and twenty-one spa types were represented, in which five MRSA showed non-type SCCmec and the remaining MRSA belonged to SCCmec type IV—where, ST1-t127 was the predominant type from all of the isolates, while ST398-t034 was the predominant type for the MRSA isolates. In this study, we found that the food-related RIF-R S. aureus may have a unique genetic background selection. However, the scenario regarding the presence of RIF-R S. aureus, especially MRSA, in retail food in China is not favorable and warrants public attention.

Profiling and identification of novel rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis clinical isolates from Pakistan

IntroductionRifampicin (RIF) is one of the most effective anti-tuberculosis first-line drugs prescribed along with isoniazid. However, the emergence of RIF resistance Mycobacterium tuberculosis (MTB) isolates is a major issue towards tuberculosis (TB) control program in high MDR TB-burdened countries including Pakistan. Molecular data behind phenotypic resistance is essential for better management of RIF resistance which has been linked with mutations in rpoB gene. Since molecular studies on RIF resistance is limited in Pakistan, the current study was aimed to investigate the molecular data of mutations in rpoB gene behind phenotypic RIF resistance isolates in Pakistan. MethodA total of 322 phenotypically RIF-resistant isolates were randomly selected from National TB Reference Laboratory, Pakistan for sequencing while 380 RIF resistance whole-genome sequencing (WGS) of Pakistani isolates (BioProject PRJEB25972), were also analyzed for rpoB mutations. ResultAmong the 702 RIF resistance samples, 675 (96.1%) isolates harbored mutations in rpoB in which 663 (94.4%) were detected within the Rifampicin Resistance Determining Region (RRDR) also known as a mutation hot spot region, including three novel. Among these mutations, 657 (97.3%) were substitutions including 603 (89.3%) single nucleotide polymorphism, 49 (7.25%) double and five (0.8%) triple. About 94.4% of Phenotypic RIF resistance strains, exhibited mutations in RRDR, which were also detectable by GeneXpert. ConclusionMutations in the RRDR region of rpoB is a major mechanism of RIF resistance in MTB circulating isolates in Pakistan. Molecular detection of drug resistance is a faster and better approach than phenotypic drug susceptibility testing to reduce the time for transmission of RIF resistance strains in population. Such insights will inform the deployment of anti-TB drug regimens and disease control tools and strategies in high burden settings, such as Pakistan.

Rarity of rpoB Mutations Outside the Rifampicin Resistance-Determining Region of Mycobacterium tuberculosis Isolates from Patients Responding Poorly to First-Line Tuberculosis Regimens in Beijing, China: A Retrospective Study

BackgroundEarly and accurate diagnosis of rifampicin (RIF)-resistant Mycobacterium tuberculosis (MTB) is essential for controlling community spread of drug-resistant tuberculosis (TB). In order to discover mutations residing outside the rifampicin resistance-determining region (RRDR) of the MTB rpoB gene, we conducted this retrospective study.MethodsWe retrospectively screened patient records to obtain Xpert MTB/RIF assay results for patients who received care at the Beijing Chest Hospital from 2016 to 2019 in order to identify subjects who met study selection criteria. Stored frozen patient isolates were cultured, harvested, and then subjected to drug susceptibility testing. Concurrently, entire rpoB gene DNA of each isolate was amplified and then sequenced to reveal rpoB mutations.ResultsOverall, 104 RIF-susceptible tuberculosis patients who were tested using the Xpert MTB/RIF assay also had poor first-line regimen treatment responses. Isolates obtained from these cases included 101 MTB isolates that possessed wild-type rpoB allelic profiles, as demonstrated using Sanger sequencing. However, sequences from the other three isolates confirmed that rpoB of one isolate harbored a mutation encoding the amino acid substitution Ile491Phe and that rpoB genes of two isolates contained a mutation encoding the amino acid substitution Ser450Leu.ConclusionOur data demonstrated that mutations found outside the RRDR of MTB rpoB are rare in Beijing, China, indicating that World Health Organization-approved molecular diagnostics are generally suitable for diagnosing RIF resistance.

In silico mutational analysis in RNA polymerase β subunit (rpoB) gene of rifampicin-resistant in Mycobacterium tuberculosis from Malaysia

Tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis (MTB) and remains as a key public health problem worldwide. Most of MTB clinical strains are resistant to rifampicin (RIF), the first-line anti-tuberculosis drug. RIF resistance to MTB is due to mutations that mainly found in RIF resistance-determining region (RRDR) in drug target gene, RNA polymerase β subunit (rpoB). Therefore, the aim of the study is to extend the identification of variants in rpoB gene and to elucidate the effect of variants to the RIF resistance. Four of the strains, MTBR1/09, MTBR2/09, MTBR3/09 and MTB221/11 were subjected to drug susceptibility test (DST). All of the strains sequenced and submitted to the National Center for Biotechnology Information Sequence Read Archive were analyzed to identify the variants in the rpoB gene. The identified new variants were then subjected to docking to examine the drug-protein interactions. DST analysis revealed MTBR1/09, MTBR2/09 and MTBR3/09 were resistant to the RIF drug, while MTB221/11 was a presumptive susceptible strain. Two new variants were observed, the deletion (Phe433_Met434delinsLeu in MTBR1/09) and missense (Lys37Arg in MTBR3/09) variants. Meanwhile, the His445Leu, Ser450Leu, Asp103Asp, Ala1075Ala were reported variants. Docking of RIF to MTBR1/09 and MTBR3/09 mutant models revealed the RIF bound to the RIF binding site at different binding affinity and conformation. Concurrently, the new variants caused the RIF to bind to the different active site and neighboring residues. Findings from DST and docking analyses indicate that new variants potentially disturb the RIF inhibition in RpoB mutant proteins, and thus might be responsible to cause the RIF resistance.

Inactivation of UmuC Protein Significantly Reduces Resistance to Ciprofloxacin and SOS Mutagenesis in Escherichia coli Mutants Harboring Intact umuD Gene

Background: Ciprofloxacin induces SOS response and mutagenesis by activation of UmuD’2C (DNA polymerase V) and DinB (DNA polymerase IV) in Escherichia coli, leading to antibiotic resistance during therapy. Inactivation of DNA polymerase V can result in the inhibition of mutagenesis in E. coli. Objectives: The aim of this research was to investigate the effect of UmuC inactivation on resistance to ciprofloxacin and SOS mutagenesis in E. coli mutants. Methods: Ciprofloxacin-resistant mutants were produced in a umuC- genetic background in the presence of increasing concentrations of ciprofloxacin. The minimum inhibitory concentration of umuC-mutants was measured by broth dilution method. Alterations in the rifampin resistance-determing region of rpoB gene were assessed by PCR amplification and DNA sequencing. The expression of SOS genes was measured by quantitative real-time PCR assay. Results: Results showed that despite the induction of SOS response (overexpression of recA, dinB, and umuD genes) following exposure to ciprofloxacin in E. coli umuC mutants, resistance to ciprofloxacin and SOS mutagenesis significantly decreased. However, rifampicin-resistant clones emerged in this genetic background. One of these clones showed mutations in the rifampicin resistance-determining region of rpoB (cluster II). The low mutation frequency of E. coli might be associated with the presence and overexpression of umuD gene, which could somehow limit the activity of DinB, the location and type of mutations in the β subunit of RNA polymerase. Conclusions: In conclusion, for increasing the efficiency of ciprofloxacin against Gram-negative bacteria, use of an inhibitor of umuC, along with ciprofloxacin, would be helpful.

Mycobacterium tuberculosis Complex Mutations in Drug Resistant Clinical Isolates from Southwest Mexico

Mutations in target genes have been described in Mycobacterium tuberculosis Complex (MTBc) drug resistant isolates worldwide. In Mexico, not enough information has been reported in this concern. The aim of this study was to characterize mutations related to resistance to first line drugs in MTBc isolates from Oaxaca, Mexico. MTBc isolates were identified in clinical samples from Tuberculosis (TB) patients. Susceptibility to isoniazid, rifampin, ethambutol, streptomycin and pyrazinamide was tested through nitrate reductase assay. PCR based analysis and sequencing were employed to characterize mutations in katG, inhA, rpoB, embB, rrs, rpsL and pncA genes. Mutations in katG and the promoter of the mabA-inhA operon were found in isoniazid resistant isolates. Sequence analysis of Rifampin Resistance-Determining Region in the rpoB gene showed novel mutations along this region besides mutations at codons 516, 526 and 531. Polymorphisms at codon 306 embB gene were found in ethambutol resistant isolates. Frequent mutations associated to resistance to streptomycin were characterized in rrs and/or rpsL genes. pncA analysis showed variable number of mutations in resistant and susceptible pyrazinamide isolates. Most frequent mutations related to resistance to first line antituberculous drugs were identified in phenotypically resistant MTBc isolates. New mutations were characterized in rpoB, rrs and rpsL genes.

RpoB gene mutations in rifampin-resistant Mycobacterium tuberculosis isolates from rural areas of Zhejiang, China.

ObjectiveThe aim was to analyze genetic mutations in the rpoB gene of rifampin-resistant Mycobacterium tuberculosis isolates (RIFR-MTB) from Zhejiang, China.MethodsWe prospectively analyzed RIFR-associated mutations in 13 rural areas of Zhejiang. Isolates were subjected to species identification, phenotype drug susceptibility testing (DST), DNA extraction, and rpoB gene sequencing.ResultsA total of 103 RIFR isolates were identified by DST (22 RIFR only, 14 poly-drug resistant, 49 multidrug resistant, 13 pre-extensively drug resistant [pre-XDR], and 5 extensively drug resistant [XDR]) from 2152 culture-positive sputum specimens. Gene sequencing of rpoB showed that the most frequent mutation was S450L (37.86%, 39/103); mutations P280L, E521K, and D595Y were outside the rifampicin resistance-determining region (RRDR) but may be associated with RIFR. Mutations associated with poly-drug resistant, pre-XDR, and XDR TB were mainly located at codon 445 or 450 in the RRDR.ConclusionsThe frequency of rpoB RRDR mutation in Zhejiang is high. Further studies are needed to clarify the relationships between RIFR and the TTC insertion at codon 433 in the RRDR and the P280L and D595Y mutations outside the RRDR.

Performance Evaluation of Xpert MTB/RIF Assay and Prevalence of rpoB Gene Mutation in <em> Mycobacterium Tuberculosis</em> in Clinical Isolates Quetta, Pakistan: A Cross Sectional Retrospective Cohort Study

With the use of Xpert assay, the detection mechanism of tuberculosis has completely transformed. Concurrently detecting mycobacterium and its resistance to rifampin (rif) which is also one of the primary marker for MDR-TB in addition to its surrogate activity against MTB in first line drugs. At large, resistance of mycobacterium strains against rifampicin is caused by rpoB gene mutations about 96.1 % globally while these mutations are generally situated at a site of 507-533rd residuals of amino acid in the rpoB gene of Mycobacterium, this region is also termed as Rifampicin resistance determining region (RRDR). In this study, we determined the mutations of various rpoB genes in different probes of 81 bp of RRD Region. A total of 6353 different specimens obtained at provincial reference laboratory of Fatima Jinnah general and chest hospital, Quetta (FJHQ), between Jan 2016 and Apr 2018 were examined by Xpert MTB/RIF assay. Altogether positive and negative samples for MTB were further examined with the light emitting diode (LED) fluorescence microscope. Pearson Chi square test was used to find out the associations between probe type and gender, treatment history and age group of the patients. Out of 6353 specimens 1297 MTB positive cases were detected by Xpert assay including 184 samples having both mycobacterium and RIF-resistance advised by mutations in 81 bp-RRD Region of rpoB gene which is further divided into five different probes. Further LED fluorescence microscope detected and confirmed 327 positive MTB samples out of 1297 specimens of Xpert assay. The associated probes mutation for rifampicin resistance were as follow: E (98/184), D (23/184), B (15/184), C (5/184), A (3/184), two probe mutation (5/184) in D&E (3/184) and C&D (2/184) and all probe mutation (35/184). The percentage of rpoB gene mutation in the studied population was 14.187 %. The commonest mutation associated with rpoB gene was in Probe E (53.2608%) also termed as 531 and 533 codons, additionally two probe mutation (2.7173%) in D&E (1.6304%) and C&D (1.0869%) and all probe mutation (19.0217%) were also detected that has never been detected before globally.

Compensatory effects of M. tuberculosis rpoB mutations outside the rifampicin resistance-determining region

ABSTRACT Mycobacterium tuberculosis has been observed to develop resistance to the frontline anti-tuberculosis drug rifampicin, primarily through mutations in the rifampicin resistance-determining region (RRDR) of rpoB. While these mutations have been determined to confer a fitness cost, compensatory mutations in rpoA and rpoC that may enhance the fitness of resistant strains have been demonstrated. Recent genomic studies identified several rpoB non-RRDR mutations that co-occurred with RRDR mutations in clinical isolates without rpoA/rpoC mutations and may confer fitness compensation. In this study, we identified 33 evolutionarily convergent rpoB non-RRDR mutations through phylogenomic analysis of public genomic data for clinical M. tuberculosis isolates. We found that none of these mutations, except V170F and I491F, can cause rifampin resistance in Mycolicibacterium smegmatis. The compensatory effects of five representative mutations across rpoB were evaluated by an in vitro competition assay, through which we observed that each of these mutations can significantly improve the relative fitness of the initial S450L mutant (0.97–1.08 vs 0.87). Furthermore, we observed that the decreased RNAP transcription efficiency introduced by S450L was significantly alleviated by each of the five mutations. Structural analysis indicated that the fitness compensation observed for the non-RRDR mutations might be achieved by modification of the RpoB active centre or by changes in interactions between RNAP subunits. Our results provide experimental evidence supporting that compensatory effects are exerted by several rpoB non-RRDR mutations, which could be utilized as additional molecular markers for predicting the fitness of clinical rifampin-resistant M. tuberculosis strains.

Prediction of rifampicin resistance beyond the RRDR using structure-based machine learning approaches

Rifampicin resistance is a major therapeutic challenge, particularly in tuberculosis, leprosy, P. aeruginosa and S. aureus infections, where it develops via missense mutations in gene rpoB. Previously we have highlighted that these mutations reduce protein affinities within the RNA polymerase complex, subsequently reducing nucleic acid affinity. Here, we have used these insights to develop a computational rifampicin resistance predictor capable of identifying resistant mutations even outside the well-defined rifampicin resistance determining region (RRDR), using clinical M. tuberculosis sequencing information. Our tool successfully identified up to 90.9% of M. tuberculosis rpoB variants correctly, with sensitivity of 92.2%, specificity of 83.6% and MCC of 0.69, outperforming the current gold-standard GeneXpert-MTB/RIF. We show our model can be translated to other clinically relevant organisms: M. leprae, P. aeruginosa and S. aureus, despite weak sequence identity. Our method was implemented as an interactive tool, SUSPECT-RIF (StrUctural Susceptibility PrEdiCTion for RIFampicin), freely available at https://biosig.unimelb.edu.au/suspect_rif/.

Molecular diagnosis, genetic diversity and drug sensitivity patterns of Mycobacterium tuberculosis strains isolated from tuberculous meningitis patients at a tertiary care hospital in South India.

Tuberculous meningitis (TBM) is the most severe form of Mycobacterium tuberculosis (Mtb) infection in humans and is a public health concern worldwide. We evaluated the performance of GeneXpert MTB/RIF (GeneXpert) for the diagnosis of TBM. In addition, genetic diversity and drug susceptibility profiling of Mtb strains isolated from TBM patients were also investigated. A total of 293 TBM suspected cerebrospinal fluid (CSF) samples were collected and subjected to GeneXpert and Mycobacterial Growth Indicator Tube (MGIT 960) culture, respectively. Sensitivity and specificity of GeneXpert was 72.7% and 98.5%, respectively by using MGIT 960 as a gold standard (GeneXpert (n = 20, 6.8%) vs MGIT 960 (n = 22, 7.5%)). All Mtb positive cultures were subjected to 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing, Line probe assay (LPA) and MGIT 960- Drug Susceptibility Testing (DST). The rpoB gene was amplified and sequenced for selected isolates. Among our TBM patients, East African Indian (EAI) lineage (n = 16, 72.7%) was most predominant followed by Beijing (n = 3, 13.6%), S-family (n = 2, 9.1%) and Delhi/CAS (n = 1, 4.5%). Three Mtb strains were found to be Isoniazid (INH) resistant by MGIT 960; however LPA revealed that two strains were INH resistant and one strain was multi drug resistant (MDR) (Resistant to Isoniazid and Rifampicin (RIF)). We identified rifampicin resistant isolate with the mutation D516F in rifampicin resistance-determining region (RRDR) and observed discordant results between LPA, GeneXpert and MGIT 960. In addition, GeneXpert showing false RIF resistance was identified (no mutation in RRDR). We conclude that GeneXpert is useful for the diagnostic confirmation of TBM; however a GeneXpert negative sample should be subjected to MGIT 960 culture or LPA to rule out TBM. EAI lineage was the most predominant among TBM patients in South India and associated with drug resistance. The discordance between GeneXpert, MGIT 960 and LPA with respect to rifampicin resistance has to be ruled out to avoid TB treatment failure or relapse.

Rapid detection of rifampicin-resistant Mycobacterium tuberculosis, based on isothermal DNA amplification and DNA chromatography

Rapid and easy detection of nucleotide point mutations in bacterial pathogens associated with drug resistance is essential for the proper use of antimicrobials. Here, we developed a rapid and simple method for the detection of mutations using Loop-mediated isothermal amplification (LAMP) combined with the single-tag hybridization (STH) chromatographic printed array strips (PAS) method. This procedure is able to detect four mutations (C1349 T, A1295C, G1303 T, A1304 T) in Rifampicin Resistance Determining Region (RRDR) of rifampicin-resistant Mycobacterium tuberculosis (RR-TB), simultaneously.LAMP reactions contained a LAMP primer and eight allele-specific primers for each mutation. The allele-specific primers products were detected by nucleic acid chromatography using PAS. Four detection lines were detected there, one of which was detected at different positions depend on the wild type and the mutant type. We carried out the four mutations detection using 31 genomic DNA (2 A1295T, 1 G1303 T, 6 A1304 T, 22 C1349 T) from clinical isolate. The mutations have been confirmed by sequence analysis. The detection results were completely consistent with the sequence analysis. In the present study, four mutations could be detected, but only 60% of RR-TB could be detected with these four. It is expected that the detection rate will increase by adding more mutant primers.The combined LAMP and STH chromatographic PAS method is a simple and rapid method for detecting point mutations in clinical isolates as a point-of-care testing (POCT) technique. In addition, it does not require special equipment and can meet the demand in areas where drug-resistant bacteria are endemic, such as developing countries.

Molecular Analysis of Rifampicin Resistance Conferring Mutations in Mycobacterium tuberculosis

Mycobacterium tuberculosis resistance to rifampicin is mainly mediated through mutations in the rpoB gene. The effects of rpoB mutations are relieved by secondary mutations in rpoA or rpoC genes. This study aims to identify mutations in rpoB, rpoA, and rpoC genes of Mycobacterium tuberculosis isolates and clarify their contribution to rifampicin resistance. Seventy isolates were identified by acid-fast bacilli smear, Genexpert assay, and growth on Lowenstein Jensen medium. Drug susceptibility, testing was performed by the proportional method. DNA extraction, PCR, and sequencing were accomplished for the entire rpoA, rpoB, and rpoC genes. Twenty-three isolates (32.85%) showed resistance to rifampicin by either proportion method or Genexpert assay. Sequence analysis of the rpoB gene revealed fourteen different mutation patterns. Inside the rifampicin resistance determining region (RRDR), codons: S531L, D516V were highly mutated with frequencies of (21.73%, 17.39%) respectively. Outside the RRDR, there were nine different types of mutations, and M479L was the most prevalent one. Out of 23 RIF resistant isolates, seven isolates (30.43%) carried mutations in the rpoA gene, and twelve isolates (52.17%) harbored a mutation in rpoC. Most of the mutations were identified for the first time in this study. The current study demonstrated that mutations in rpoB, rpoA, and rpoC contributed to RIF resistance in Mycobacterium tuberculosis and this new finding may be relevant to realize how compensatory mutations in the rpoA and rpoC genes restore the fitness cost caused by rifampin resistance-conferring mutations in rpoB.

Distribution and Frequency of rpoB Mutations Detected by Xpert MTB/RIF Assay Among Beijing and Non-Beijing Rifampicin Resistant Mycobacterium tuberculosis Isolates in Bangladesh.

BackgroundRifampicin resistance (RR) is a key indicator of multidrug-resistant tuberculosis (MDR-TB) and 95% of the RR is associated with the mutation in the 81-bp rifampicin resistance determining region (RRDR) of the rpoB gene of Mycobacterium tuberculosis complex (MTBC). The Xpert MTB/RIF (Xpert) assay uses five overlapping molecular beacon probes (A-E) complementary to RRDR region that detect MTBC and mutations associated with RR. The objective of the study was to investigate the distribution and frequency of mutations detected by Xpert assay among Beijing and non-Beijing RR-TB isolates.MethodsA total of 205 randomly selected RR-TB specimens detected by Xpert assay were included in this study. A portion of specimens was further subjected to culture, MTBDRplus test and the positive culture isolates were genotyped by spoligotyping.ResultsWe found that the most frequent mutation occurred at probe E (S531L) binding region in both Beijing and non-Beijing isolates (61.9% and 66.9%, respectively). The Beijing family had higher mutation rates than non-Beijing (19.0% vs 12.4%) at probe B (D516V) while the non-Beijing family had higher mutations at probe D (H526D or H526Y) than the Beijing (13.2% vs 10.7%) family. Mutations at probes Aand C were less common in both Beijing and non-Beijing isolates. There was no significant difference (P=0.36) in the occurrence of mutations at different probes between Beijing and non-Beijing isolates.ConclusionsThe study results revealed that the most frequent mutation occurs in the region of probe E and the least common mutations at probe A and C among both Beijing and non-Beijing RR-TB cases. This first insight into the probe mutation variation and frequencies among the RR-TB cases in Bangladesh forms the baseline information for further investigation.

Helicobacter pylori Infections in the Bronx, New York: Surveying Antibiotic Susceptibility and Strain Lineage by Whole-Genome Sequencing.

The emergence of drug resistance in Helicobacter pylori has resulted in a greater need for susceptibility-guided treatment. While the alleles associated with resistance to clarithromycin and levofloxacin have been defined, there are limited data regarding the molecular mechanisms underlying resistance to other antimicrobials. Using H. pylori isolates from 42 clinical specimens, we compared phenotypic and whole-genome sequencing (WGS)-based detection of resistance. Phenotypic resistance correlated with the presence of alleles of 23S rRNA (A2142G/A2143G) for clarithromycin (kappa coefficient, 0.84; 95% confidence interval [CI], 0.67 to 1.0) and gyrA (N87I/N87K/D91Y/D91N/D91G/D99N) for levofloxacin (kappa coefficient, 0.90; 95% CI, 0.77 to 1.0). Phenotypic resistance to amoxicillin in three isolates correlated with mutations in pbp1, pbp2, and/or pbp3 within coding regions near known amoxicillin binding motifs. All isolates were phenotypically susceptible to tetracycline, although four bore a mutation in 16S rRNA (A926G). For metronidazole, nonsense mutations and R16H substitutions in rdxA correlated with phenotypic resistance (kappa coefficient, 0.76; 95% CI, 0.56 to 0.96). Previously identified mutations in the rpoB rifampin resistance-determining region (RRDR) were not present, but 14 novel mutations outside the RRDR were found in rifampin-resistant isolates. WGS also allowed for strain lineage determination, which may be important for future studies in associating precise MICs with specific resistance alleles. In summary, WGS allows for broad analyses of H. pylori isolates, and our findings support the use of WGS for the detection of clarithromycin and levofloxacin resistance. Additional studies are warranted to better define mutations conferring resistance to amoxicillin, tetracycline, and rifampin, but combinatorial analyses for rdxA gene truncations and R16H mutations have utility for determining metronidazole resistance.