Enriched whole genome sequencing identified compensatory mutations in the RNA polymerase gene of rifampicin-resistant Mycobacterium leprae strains

Infection and Drug Resistance, Jan 2018

Enriched whole genome sequencing identified compensatory mutations in the RNA polymerase gene of rifampicin-resistant Mycobacterium leprae strains Mallika Lavania,1 Itu Singh,1 Ravindra P Turankar,1 Anuj Kumar Gupta,2 Madhvi Ahuja,1 Vinay Pathak,1 Utpal Sengupta,1 1Stanley Browne Laboratory, The Leprosy Mission Trust India, TLM Community Hospital Nand Nagari, 2Agilent Technologies India Pvt Ltd, Jasola District Centre, New Delhi, India Abstract: Despite more than three decades of multidrug therapy (MDT), leprosy remains a major public health issue in several endemic countries, including India. The emergence of drug resistance in Mycobacterium leprae (M. leprae) is a cause of concern and poses a threat to the leprosy-control program, which might ultimately dampen the achievement of the elimination program of the country. Rifampicin resistance in clinical strains of M. leprae are supposed to arise from harboring bacterial strains with mutations in the 81-bp rifampicin resistance determining region (RRDR) of the rpoB gene. However, complete dynamics of rifampicin resistance are not explained only by this mutation in leprosy strains. To understand the role of other compensatory mutations and transmission dynamics of drug-resistant leprosy, a genome-wide sequencing of 11 M. leprae strains – comprising five rifampicin-resistant strains, five sensitive strains, and one reference strain – was done in this study. We observed the presence of compensatory mutations in two rifampicin-resistant strains in rpoC and mmpL7 genes, along with rpoB, that may additionally be responsible for conferring resistance in those strains. Our findings support the role for compensatory mutation(s) in RNA polymerase gene(s), resulting in rifampicin resistance in relapsed leprosy patients. Keywords: leprosy, rifampicin resistance, compensatory mutations, next generation sequencing, relapsed, MDT, India

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Enriched whole genome sequencing identified compensatory mutations in the RNA polymerase gene of rifampicin-resistant Mycobacterium leprae strains

Infection and Drug Resistance Enriched whole genome sequencing identified compensatory mutations in the Rna polymerase gene of rifampicin-resistant Mycobacterium leprae strains Mallika lavania 1 Itu singh 1 Ravindra P Turankar 1 anuj Kumar gupta 0 Madhvi ahuja 1 Vinay Pathak 1 Utpal sengupta 1 0 agilent Technologies India Pvt ltd, Jasola District centre , n ew Delhi , India 1 stanley Browne laboratory, The leprosy Mission Trust India, TlM community hospital n and n agari Despite more than three decades of multidrug therapy (MDT), leprosy remains a major public health issue in several endemic countries, including India. The emergence of drug resistance in Mycobacterium leprae (M. leprae) is a cause of concern and poses a threat to the leprosy-control program, which might ultimately dampen the achievement of the elimination program of the country. Rifampicin resistance in clinical strains of M. leprae are supposed to arise from harboring bacterial strains with mutations in the 81-bp rifampicin resistance determining region (RRDR) of the rpoB gene. However, complete dynamics of rifampicin resistance are not explained only by this mutation in leprosy strains. To understand the role of other compensatory mutations and transmission dynamics of drug-resistant leprosy, a genome-wide sequencing of 11 M. leprae strains - comprising five rifampicin-resistant strains, five sensitive strains, and one reference strain - was done in this study. We observed the presence of compensatory mutations in two rifampicin-resistant strains in rpoC and mmpL7 genes, along with rpoB, that may additionally be responsible for conferring resistance in those strains. Our findings support the role for compensatory mutation(s) in RNA polymerase gene(s), resulting in rifampicin resistance in relapsed leprosy patients. - 8 1 0 2 l u J 2 1 n o 7 0 2 . 6 4 . 9 5 . 7 3 y b / m o c . s s e r dapsone, and clofazimine. Rifampicin, the backbone of MDT, is a bactericidal drug, which binds to the beta subunit of the RNA polymerase encoded by rpoB and inhibits transcription. Recent reports have, however, indicated the occurrence of rifampicin resistance in several endemic areas such as Brazil, India, and People’s Republic of China.2,3,5–7 At this present stage, it is important to monitor the emergence of rifampicin-resistant mutants in endemic populations where transmission of M. leprae infection is continuing. On the other hand, resistance to dapsone has been reported since the late 1960s. Moreover, a strain showing resistance to both dapsone and rifampicin was reported in 1993.8 At present, further reports indicating the emergence of M. leprae strains resistant to multiple drugs are also emerging.2,9,10 As M. leprae cannot be cultured in vitro, molecular drug susceptibility testing, including PCR-sequencing, for determining resistant strains provides a practical alternative. At present, the rapid detection and control of such drug-resistant .vdoepww l.syoeun tsitoraninlesvieslse,sssuecnhtiaalsiInndcoiau.nAtrsiensewapcparsoeascahriengcolenptirnousiyngel,iwmhinicah/sw lan is considered a major indicator for disease transmission, there / : ttph rsoe is still a need for better measures to control leprosy. Furtherfrom ropF more, relapsed cases with drug-resistant strains could be a lodaed tnuerwe osfouelricmeionfatdioisne,aistewtirlalnbsemeisssseionnti.aTlthoerceofnodreu,cattatthhiosrjouungchcndoew innevwesctaigsaetsi oton iodfenretilfayptsheedpmatuteltrinbaocfilelxairsyte(nMceBo)fcparsiems aarnydanaldl itsna secondary resistance to anti-leprosy drugs such as rifampicin seR and dapsone. Whole genome sequencing (WGS) of M. leprae rgu directly from clinical samples is a good tool for identifying dnD mutations that cause drug resistance, especially those that are noa not known or well characterized. In biopsy samples, M. leprae itfce genome copies can vary in number, but the relative proporIn tion of bacterial DNA is minute in comparison to host DNA. Direct sequencing of mixed human and bacterial DNA does not yield significant proportions of sequence reads that map to the bacterial genome. For this reason, we used a capture probebased bacterial DNA enrichment method which selectively isolates bacterial DNA from host DNA prior to sequencing. With the help of this approach, the present study focuses on identifying compensatory mutations in genes other than, but related to, rpoB in rifampicin-resistant strains of M. leprae. Materials and methods ethical approval Ethical clearance for this study was approved by the The Leprosy Mission Trust India Ethical Committee, held on August 29, 2014, under the regulations of the Indian Council submit your manuscript | www.dovepress.com Dovepress of Medical Research. Written informed consent was obtained from all subjects before collection of biological samples. source of biopsies for Dna extraction A total of ten relapsed leprosy patients from hospitals of The Leprosy Mission Trust India (TLMTI) and one reference control strain (Br4923) from BEI Resources, USA, were included in this study. Among these ten strains, five were resistant to rifampicin, characterized by molecular methods and PCRsequencing. The remaining five strains were drug sensitive. M. leprae DNA was extracted from these ten clinical biopsy specimens using the Blood and Tissue DNA extraction kit (QIAGEN, USA). Single-nucleotide polymorphism (SNP) subtyping data was generated by PCR sequencing11 from DNA before carrying out WGS for the remaining DNA. library preparation and sequencing Target capture sequencing libraries were prepared with Illumina-compatible SureSelect QXT Library Prep Reagent Kit (Agilent Technologies, Santa Clara, CA, USA) at Genotypic Technology Pvt. Ltd., Bangalore, India. Briefly, 50 ng Qubit-quantified genomic DNA was fragmented and adapter-tagged using SureSelect QXT Enzyme (Table S1). The fragmented and adapter-tagged DNA was purified with HighPrep magnetic beads and then amplified by eight cycles of PCR. The PCR-enriched products were purified with HighPrep beads, followed by library quality control check using the Agilent 2100 Bioanalyzer. Target enrichment was undertaken according to the manufacturer’s instructions using SureSelect RNA capture baits. In-solution hybridization was done as per the SureSelect QXT hybridization method. After hybridization, the captured biotinylated probe-target hybrids were pulled down by using streptavidin-coated magnetic beads (Dynabeads MyOne Streptavidin T1, ThermoFisher Scientific, Waltham, MA, USA). The magnetic beads were washed according to the manufacturer’s instructions and resuspended in 15 lμ nuclease-free water. The captured DNA libraries were amplified by 12 cycles of PCR with the inclusion of the appropriate indexing primer for each sample. The final PCR product (sequencing library) was purified with HighPrep beads, followed by quantification by a Qubit fluorometer (ThermoFisher Scientific, MA, USA), and fragment size distribution was analyzed on an Agilent 2100 Bioanalyzer. Sequencing was carried out on an Illumina Hi-Seq 2500 instrument and the Illumina paired-end raw reads were quality checked using FastQC. Illumina raw reads were processed for adapters and trimming of low-quality bases. The processed reads were aligned with M. leprae TN reference using a Burrows–Wheeler Aligner (BWA) 0.7.5 algorithm, and the variants were identified with Samtools 1.2 and Bcftools 1.2 filtered for a read depth threshold of greater than 20, and a quality threshold of more than Q30. Filtered variants were further annotated with SnpEff to get the information on the genes, protein change, and the impact of the variation. Results Demographics and diagnosis Among the five rifampicin-resistant patients, one patient was a defaulter who took MDT for 2 months only and remaining four patients were those who relapsed after completion of a full course of MDT. One of the patients (1027) had a dual infection of leprosy and tuberculosis. The demographic and clinical details of these five resistant patients together with the five sensitive ones are mentioned in Table 1A and B. All resistant strains were characterized for drug-susceptibility testing by PCR sequencing. We found the already published rpoB mutations in three patients (507, 42, and 80) at codon positions Leu436Meth, Asp441Tyr, and Ser456Leu, except in two (1027 and 439) who had mutations at codon positions Gln442His and Ser437Pro (Table 2). These two patients did not respond to the treatment. Gln442His mutation in patient 1027 and Ser437Pro in patient 439 of rpoB gene did not confer resistance to rifampicin, which was confirmed by mouse foot pad growth earlier.12 The patient 1027 continuously visited the hospital with recurrent Type 2 reaction and other complications despite being administered thalidomide. He was later diagnosed with a pulmonary tuberculosis infection along with leprosy. He was started on anti-tuberculosis treatment (ATT) as well, but the infection was unalleviated. We tested the same specimen with Mycobacterium tuberculosis-specific rpoB primers, undertook DNA sequencing, and Patients’ identification age at diagnosis (years) sex start of MDT (month/year) completion of MDT (month/year) Relapse date RJ Classification at diagnosis at relapse Bacillary index First diagnosis Relapse clinical complication at the time of relapse B Patients’ identification age at diagnosis (years) sex start of MDT (month/year) completion of MDT (month/year) Relapse date RJ Classification at diagnosis at relapse Bacillary index First diagnosis Relapse clinical complication at the time of relapse 507 23 Male 10/2011 10/2011 08/2016 BB BB 0.66+ 2.0+ none 1,335 38 Male 11/2008 10/2009 12/2015 Bl ll 42 31 Male 05/2013 05/2014 05/2015 BT Bl 834 19 Male 2005 2007 10/2010 ll ll 3.0+ 4.0+ Infiltration with raised reddish lesions 2.0+ 2.66+ Infiltration all over the body 1.0+ 2.0+ Infiltration with Type 1 reaction 2.0+ 3.0+ Infiltration with new lesions 4.33+ 4.33+ Multiple enls on face Abbreviations: BB, borderline leprosy; Bl, borderline lepromatous; BT, borderline tuberculoid; ll, lepromatous leprosy; MDT, multidrug therapy; na, not available; RJ, Ridley–Jopling. 1027 18 Male 11/2011 11/2012 0 1/2015 Bl ll SM 40 Male 2005 2007 0 4/2017 na Bl 439 18 Male 2013 2014 0 7/2015 Bl ll na 6.0+ none 728 34 Female 2008 2009 10/2010 ll ll 80 18 Female 02/2012 02/2013 12/2015 Bl Bl 730 33 Male 04/2013 04/2014 12/2015 Bl rpoC mutations (WGS) Val517leu nM nM nM leu807Met; leu244Phe 8 1 0 2 l u J 2 1 n o 7 0 2 . 6 4 . 9 5 . 7 3 y b / m o c . s s e r found him susceptible for the TB rpoB gene. This led us to consider the possibility that other mutations at other genes play a role in conferring resistance in such cases. genetic polymorphisms Whole genome read coverage was adequately generated from the 10 M. leprae isolates, and the reference strain for comparative genomic analysis. Among these ten samples, .vdoepww l.syoeun 1bSa1Ns,7Pe9st1oanfvidaltre9ira5no8tus(t8wc.0oe%mrem)iIodnnednevtlaisfr.iieaAdns,tsti,hnwecrleeufdiosincnugose1pd0r,eo8vn3i3orpu(os9B2daa%tnad-) /w la / : ttsp rson its associated genes. After analyzing the data of the five h pe rifampicin-resistant strains, we found that two strains showed frdom roF mutation in the rpoC gene (Patient ID 1027 and 439) along dae with the rpoB gene (Table 2) as well as a non-synonymous lnow mutation in the resistance nodulation division (RND) family ode transporter efflux pump gene mmpL 7. These two genes are itsscnea tmheaiSnlNyPrsespproensesnibtlienftohrecsaeusstirnaginrse.sistance among the rest of gR Analysis of rifampicin-resistant strains revealed 1,308 ruD variants, of which there were 44 synonymous, 101 nondan synonymous, 112 intergenic, and 12 frame-shift mutations. itcon On genome comparison of all ten strains, we observed seven fIen unique SNPs. While comparing resistant strains with other sensitive strains, it revealed 16 unique SNPs that were present only in these strains (Table 3). An insertion of eight nucleotides (TTTCTTAT at position 508802) was found in ML0411, encoding the Pro-Pro-Glu (PPE) serine-rich antigen. Discussion The emergence of rifampicin resistance in M. leprae after more than 30 years of MDT is not an unexpected outcome. The occurrence of dapsone resistance also took nearly the same duration. However, the emergence of rifampicin-resistant M. leprae during the phase of elimination may cause a major setback to the public health program. Drug-resistant leprosy infection can be caused by transmission of already resistant strains (primary resistance) or by selection of resistance-conferring mutations during inadequate therapy (secondary resis172 rpoB mutations (WGS)* arg1168leu; arg505Trp Iso433ser; leu436 Met leu458Phe Tyr-cys Tyr-cys tance). Our own work2,3 has shown the presence of rifampicin, dapsone, and ofloxacin resistance cases in highly endemic areas in several states of India. M. leprae isolates resistant to single and multiple drugs have already been encountered.2,13 Although the genome of M. leprae has undergone massive gene decay,14 the rifampicin resistance determining region (RRDR) region of the organism and the associated genes are still functional. In order to monitor the transmission dynamics of drug-resistant leprosy, a comparison of M. leprae strains was carried out by WGS of strains derived from rifampicinresistant leprosy patients in this study. Rifampicin inhibits bacterial RNA synthesis by binding strongly to RNA polymerase, preventing attachment of the enzyme to DNA and thus blocking the initiation of transcription. Bacterial resistance to rifampicin is caused by mutations leading to a change in the structure of the beta subunit of RNA polymerase. Resistance to rifampicin is one of the main causes of treatment failure.15 Various studies have shown that resistance to rifampicin in M. tuberculosis as well as in M. leprae is mainly due to a single mutation in an 81-bp region in the rpoB gene.16,17 Furthermore, it has been reported earlier that there are some alternative mechanisms of rifampicin resistance in various bacterial species such as rox gene-mediated mono-oxygenation of rifampicin18 and duplication of the rpoB gene as rpoB2 in Nocardia.19 In our study, all strains belonged to the SNP subtype 1D, which is the predominant genotype prevalent in many countries such as India, Bangladesh, Nepal, Madagascar, Malawi, and the French West Indies.11,20,21 The coexistence of the two mycobacteria has been demonstrated in archaeological samples by Donoghue et al,22 who identified DNA from both tuberculosis and leprosy in the archeological samples from the Roman period to the 13th century from several sites around the world. On review of data from three leprosy referral centers in Hyderabad, India, from 2000 to 2013, Rawson et al23 identified three cases of concomitant disease. Similarly, we observed in one of our samples a patient (Pt1027) who is suffering from both TB and leprosy. Amino acid change T/M Type Probable ferredoxin-dependent glutamate synthase Probable naDh-dependent glutamate synthase Possible 10 KDA culture filtrate antigen homolog esxB (lhp) (cfp10) conserved membrane protein Probable conserved membrane protein Putative primosomal protein n’ Pria (Replication factor Y) Probable bifunctional riboflavin-specific deaminase/reductase Probable integral membrane indolylacetylinositol arabinosyltransferase emba (arabinosylindolylacetylinositol synthase) Probable integral membrane indolylacetylinositol arabinosyltransferase embc (arabinosylindolylacetylinositol synthase) Dna-directed Rna polymerase (beta’ chain) conserved transmembrane transport protein Mmpl7 Possible transcriptional regulatory protein Probable two component sensor kinase MprB Probable conserved transmembrane protein Probable alanyl-tRna synthetase alas (alanine–tRna ligase) and alaRs (alanine translase) 8 1 0 2 l u J 2 1 n o 7 0 2 . 6 4 . 9 5 . 7 3 y b / m o c . s s e r .vdoepww l.syoeun /w la /:s n ttp rso h e p from roF d e d a o l n w o d e c n a it s s e R g u r D d n a n o it c e f n I This study provides a better explanation for the presence of the rifampicin-resistance phenotype in leprosy cases in India. It has identified unique SNPs, including non-synonymous SNPs in gltB, gltD, and esxB and mutation in ctpC and mmpL7 transporter genes. The mmpL7 gene in M. tuberculosis was studied and characterized by Pasca et al.24 It encodes a protein which belongs to RND drug transporter family. The MmpL7 protein is of 920 amino acids with a predicted molecular mass of 95.1 kDa, contains 12 transmembrane domains (TMDs) having two large hydrophilic extracytoplasmic domains between TMD-1 and TMD-2 and between TMD-7 and TMD-8. All of these characteristics have been described as typical of RND efflux pumps.25 The MmpL7 protein confers a high level of resistance to isoniazid.24 However, a functional assay is required to determine whether either of these variant genes/transporters confers any degree of rifampicin resistance in M. leprae. In our study, the rpoC gene has a non-synonymous SNPs sequence in two patients – 1027 and 439. Mouse foot pad assay was done for the patient 1027 to check whether the mutation in 442 codon position was conferring resistance. In our previous study, we observed that this 442 codon mutation was not conferring resistance in mice.12 Therefore, it may be suggested that these compensatory mutations in rpoC and/or transporter genes play a role in conferring resistance in this strain. Thus, our study suggested the role of compensatory mutation in rpoC and possibly of drug transporter genes in contributing to acquired resistance in rifampicin-resistant strains in M. leprae. Acknowledgments The authors thank the Indian Council of Medical Research (Grant No. ECD/Ad-hoc/leprosy/2014-113/Fy. 14-15/19/ Delhi/NGO-ECD-I) and England and Wales Foundation (205T03) for the financial support. The authors are likewise grateful to Atul Roy and Manish Gardia for assisting us in the sample collection, and thank the superintendent and staff of TLM Hospitals for their help and assistance during the work. Disclosure The authors report no conflicts of interest in this work. submit your manuscript | www.dovepress.com Dovepress 8 1 0 2 l u J 2 1 n o 7 0 2 . 6 4 . 9 5 . 7 3 y b / m o c . s s e r from roF sM 728 1335 730 Bh Supplementary material Sample ID DNA concentration by Qubit (ng/μL) Library concentration (ng/μL) Volume Publish your work in this journal Infection and Drug Resistance is an international, peer-reviewed openaccess journal that focuses on the optimal treatment of infection (bacterial, fungal and viral) and the development and institution of preventive strategies to minimize the development and spread of resistance. The journal is specifically concerned with the epidemiology of antibiotic 1. NLEP. NLEP Annual report 2015-2016. Available from: www.nlep. nic.in. Accessed July 20 , 2017 . 2. Lavania M , Jadhav RS , Chaitanya VS , et al. 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Mallika Lavania, Itu Singh, Ravindra P Turankar, Anuj Kumar Gupta, Madhvi Ahuja, Vinay Pathak, Utpal Sengupta. Enriched whole genome sequencing identified compensatory mutations in the RNA polymerase gene of rifampicin-resistant Mycobacterium leprae strains, Infection and Drug Resistance, 2018, 169-175, DOI: 10.2147/IDR.S152082