Microbiological Research 166 (2011) 121—128

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High-resolution melting facilitates mutationscreening of rpsL gene associated withstreptomycin resistance in Mycobacteriumtuberculosis

Feifei Wanga,b,1, Hongbo Shena,1, Ming Guanc, Ying Wangb, Yun Fengb,Xinhua Wengb, Honghai Wanga,�, Wenhong Zhangb,d,��

aState Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai200433, ChinabDepartment of Infectious Disease, Huashan Hospital, Fudan University, Shanghai, ChinacCenter of Laboratory Medicine, Huashan Hospital, Fudan University, Shanghai, ChinadInstitutes of Biomedical Sciences, Fudan University, Shanghai, China

Received 9 December 2009; received in revised form 7 February 2010; accepted 7 February 2010

KEYWORDSHigh-resolution meltanalysis;Mutation;Mycobacterium tu-berculosis;rpsL gene;Streptomycin resis-tance

ee front matter & 2010micres.2010.02.001

ing author. Tel.: +86 21ding author at: Depart888123; fax: +86 21 624esses: hhwang@fudan.eequally to this work.

SummaryDrug resistance remains a serious threat to tuberculosis control worldwide. As one ofthe important first-line antitubercular agents, resistance to streptomycin (SM) andits derivatives has increased in recent years and has become one of thecharacteristics of extensively drug-resistant tuberculosis (XDR-TB). A novel rapidassay to screen for rpsL gene mutations associated with SM resistance inMycobacterium tuberculosis (M. tuberculosis), was developed using high-resolutionmelting (HRM) analysis. The HRM results of 134 SM-resistant clinical isolates and 20SM-susceptible clinical isolates of M. tuberculosis for rpsL gene mutations wereperfectly matched with those of DNA sequencing. SM resistance was highlyassociated with rpsL mutations in M. tuberculosis. HRM technique represented aninexpensive, highly sensitive and high-throughput method to facilitate the screeningof large numbers of clinical samples for epidemiological studies of drug-resistance ofM. tuberculosis, especially in developing countries.& 2010 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights reserved.

65643777; fax: +86 21 65648376.ment of Infectious Disease, Huashan Hospital, Fudan University, Shanghai, 200040, China.89015.du.cn (H. Wang), zhangwenhong@fudan.edu.cn (W. Zhang).

F. Wang et al.122

Introduction

Tuberculosis (TB) remains one of the leading causesof death from infectious diseases, with about 9.2million new cases of TB and 1.7 million deathsworldwide annually (WHO 2008). It is estimated thatone-third of the world’s population is infected withMycobacterium tuberculosis. Drug resistance remainsa serious threat to tuberculosis (TB) control world-wide (Aziz and Wright 2005; Matteelli et al. 2007;Riccardi et al. 2009; Jassal and Bishai 2009).

Streptomycin (SM) has shown to be an effectiveantitubercular drug since 1944 (Blanchard 1996).Recently, the emergence of strains of M. tuberculosisdisplaying resistance to some or all of the major anti-tuberculosis drugs (isoniazid, rifampicin, ethambutol,pyrazinamide and fluoroquinolones) has led torenewed interest in streptomycin and its derivatives,kanamycin and amikacin. Two genes, rpsL and rrs,encoding the ribosomal protein S12 and the 16S rRNA,are known to be responsible for SM resistance(Douglass and Steyn 1993; Finken et al. 1993). InChina, the frequency of rpsL and rrs mutations inSM-resistant clinical isolates is high because strepto-mycin has been used for many years as one of the first-line drugs of regular antitubercular regimens. Strepto-mycin acts by binding to the 30S ribosomal subunit,thereby interfering with polypeptide synthesis byinhibiting translation (Springer et al. 2001). Mutationsin rpsL at codons 43 and 88 are more common andcorrelate with high levels of resistance (Katsukawaet al. 1997; Sun et al. 2009). Using a novel rapid assay,this study was designed to investigate rpsL mutationsin SM-resistant clinical isolates and SM-susceptibleclinical isolates from China.

Currently, detection of mutations in drug-resis-tant strains of M. tuberculosis relies upon timeconsuming and labor intensive procedures such asPCR-RFLP, PCR-SSCP and molecular beacons. Thecomplexity of these methods and the need formultiple steps to perform them greatly increase therisk of cross-contamination and thereby misdiagno-sis. Recently, a novel technique, high-resolutionmelting (HRM) has been investigated for thedetection of point mutations, single-nucleotidepolymorphism (SNP), internal tandem duplications,simultaneous mutation scanning and genotyping inbacteriology, cancer research and human plateletantigens. This method requires only the usualunlabeled primers and a dsDNA binding dye and isa closed-tube, thereby minimizing cross-contam-ination (Krypuy et al. 2006; Liew et al. 2006;Jeffery et al. 2007; Montgomery et al. 2007;Ruppitsch et al. 2008; Millat et al. 2009; Pietzkaet al. 2009).

The aim of our study was to develop and evaluateHRM curve analysis as a suitable PCR-based screen-ing method to screen rpsL gene mutations asso-ciated with SM resistance in M. tuberculosis. Wereported an optimized protocol for scanning therpsL gene of M. tuberculosis by HRM analysis usingthe Rotor-Gene 6000 (Corbett Life Science). To ourknowledge, this is the first report to describe HRManalysis of mutations of rpsL gene associated withresistance of M. tuberculosis to streptomycin in alarge number of clinical isolates.

Materials and Methods

M. tuberculosis strains and DNA extraction

The reference strains (H37Rv, MU1, MU2), 134SM-resistant clinical isolates and 20 SM-susceptibleclinical isolates of M. tuberculosis were obtainedfrom Shanghai Pulmonary Disease Hospital in China.Genomic DNA was extracted using QIAamp DNA Minikit (QIAGEN Inc., Germany) according to themanufacturer’s instructions. Two reference strains(MU1, MU2) with characterized mutations of rpsLgene were previously detected by direct sequen-cing analysis. MU1 had a mutation (AAG-AGG,Lys-Arg) at codon 43. MU2 had a mutation (AAG-AGG, Lys-Arg) at codon 88. rpsL gene wassequenced directly in 20 SM-susceptible clinicalisolates and 104 strains from 134 SM-resistantclinical isolates of M. tuberculosis, 44 isolates ofwhich were low-resistant isolates and the other 60isolates were found to be high-resistant to strepto-mycin by phenotypic drug susceptibility testing.Resistance to SM in clinical isolates of M. tubercu-losis was determined according to the standards ofNCCLS (2004). The 104 strains from 134 SM-resistant clinical M. tuberculosis isolates and 20SM-susceptible clinical isolates were detected byHRM analysis comparing with direct sequencing.The remaining 30 SM-resistant clinical isolates(numbers: S1–S30) were detected by HRM analysisas a validation test in a blind study.

Amplification of rpsL gene and directsequencing

Amplification of rpsL gene was performed using theprimers 50-CAAACAGAACGTGAAAG-30 and 50-CGTA-GACCGGGTCGTTG-30. The PCR reaction mix wasprepared using KOD-Plus (TOYOBO) containing thefollowing components per reaction: DNA template(1 mL), 10� PCR buffer for KOD-Plus (5 mL), 25 mM ofMgSO4 (2 mL), 10 mM of each primer (2 mL), 2 mM of

Mutation screening of rpsL gene in M. tuberculosis 123

dNTPs (5 mL), 1 mL of KOD DNA polymerase (1 U/mL)and PCR grade water to a final volume of 50 mL. PCRwas performed as follows: 94 1C for 5 min; 94 1C for45 s, 64 1C for 40 s, 72 1C for 30 s for 35 cycles; andfinally, 72 1C for 10 min. PCR products were sent toInvitrogen for DNA sequencing. The sequences of allsamples were compared using MegAlignTM software(DNASTAR Inc., Madison, WI).

Amplification of amplicons and HRM assays

Two pair of primers were designed to amplify a199 base pair amplicon (rpsL-a) and a 235 base pairamplicon (rpsL-b) spanning the target region ofrpsL gene associated with SM resistance, respec-tively. Primers for the 199 base pair (bp) ampliconwith codon 43 were 50-AGAAAGCCGGTAGATGCCAA-30 (rpsL-a F) and 50-CTCGACCTGACTCGTCAACT-30 (rpsL-a R). In addition, primers for the 235bp amplicon with codon 88 were 50-GGCGCTTCGGAAGGTTG-30 (rpsL-b F) and 50-TCAGCCCTTCTCTTTCTT-30 (rpsL-b R). The interca-lating dye used was Eva GreenTM (Biotium Inc.,USA). The reaction mixture was made up usingKOD-Plus (TOYOBO) and consisted of genomic DNA(0.5 mL), 10� PCR buffer for KOD-Plus (2 mL),25 mM of MgSO4 (0.8 mL), 10 mM of each primers(rpsL-a 0.5 mL, or rpsL-b 0.8 mL), 2 mM of dNTPs(2 mL), Eva GreenTM 20� (1 mL), 0.5 U of KOD DNApolymerase and PCR grade water in a volume of20 mL. The thermal cycling conditions were 94 1Cfor 5 min followed by 40 cycles with denaturationstep at 94 1C for 30 s, annealing at 68 1C (rpsL-a) or65 1C (rpsL-b) for 30 s, and extension at 72 1C for30 s. All samples were tested in duplicate.

HRM analysis of 104 clinical isolates was per-formed using the Rotor-GeneTM 6000 analyser(Corbett Life Science). A pre-melt hold at 60 1Cfor 30 s was allowed prior to HRM. Melting-curvedata were generated by increasing the temperaturefrom 72 to 95 1C at 0.1 1C/s and recordingfluorescence in the green channel. HRM curveanalysis was performed using Rotor-Gene 6000Series Software (Version 1.7). The software was

Table 1. Type of rpsL mutation in 104 isolates of SM-resist

Type of rpsL mutation SM-resistant M. tuberculosis (104

Low-resistant isolates (44) Hig

Numbers Ratio (%) Nu

Codon 43 (AAG-AGG) 16 16/44 (36.4%) 43Codon 88 (AAG-AGG) 15 15/44 (34.1%) 8No mutation 13 13/44 (29.5%) 9

used to calculate the derivative of the intensity offluorescence at different temperatures (dF/dT).The reference strains (H37Rv, MU1, MU2) wereincluded in each run to facilitate inter-run compar-isons. All samples were tested in duplicate toensure reproducibility of the melt curves. The HRMcurves were derived by first selecting two normal-isation regions, one occurring prior to the meltingof the double stranded product and one followingcomplete separation of the two strands.

Results

rpsL gene mutations in clinical isolates

rpsL gene was sequenced for reference strainH37Rv along with 104 SM-resistant clinical isolatesand 20 SM-susceptible clinical isolates. The resultsof DNA sequencing showed that 78.8% (82/104) ofSM-resistant isolates harbored rpsL mutations,while codon 43 dominated. 56.7% (59/104) isolateshad a mutation (AAG-AGG, Lys-Arg) at codon 43.Comparatively, codon 88 mutation played a minorrole and 22.1% (23/104) isolates had a mutation(AAG-AGG, Lys-Arg) at codon 88. However, nomutation of rpsL gene was found in the remaining21.2% (22/104) of the SM-resistant isolates bydirect sequencing method (Table 1). All of the 20SM-susceptible isolates had wild-type rpsL.

HRM analysis comparing with directsequencing

We optimized the PCR reactions to ensure thatthere were no primer dimers or non-specificproducts. The standard melt analysis softwaremodule showed there was a single peak indicativeof a single product in rpsL-a amplicon and rpsL-bamplicon, respectively (Figure 1(A) and (B)).

We used reference strains (H37Rv, MU1, MU2) induplicate for each run. rpsL gene mutations of teststrains were then determined by comparing theHRM curves with the reference strains using Rotor-

ant M. tuberculosis.

)

h-resistant isolates (60) Total isolates (104)

mbers Ratio (%) Numbers Ratio (%)

43/60 (71.7%) 59 59/104 (56.7%)8/60 (13.3%) 23 23/104 (22.1%)9/60 (15.0%) 22 22/104 (21.2%)

Figure 1. Melt analysis of rpsL amplicons. The standard melt analysis software module showed there was a single peakindicative of a single product in rpsL-a amplicon and rpsL-b amplicon, respectively. (A) The standard melt analysis ofrpsL-a amplicon. (B) The standard melt analysis of rpsL-b amplicon.

F. Wang et al.124

Gene 6000 software. The partial results of HRManalysis for rpsL-a amplicon and rpsL-b ampliconwere shown in Figure 2(A)–(D). The ‘‘differencegraph’’ was generated by selecting the H37Rvreference strain melting profile as the wild type.All wild types were then normalised to zero and anydeviations (i.e. mutant type) from this referencegenotype were highlighted in the difference graphas a positive curve. It showed a clear separationbetween the wild type and the mutant type using a‘‘difference graph’’. The results of HRM analysiswere completely consistent with those of DNAsequencing. By performing HRM analysis on isolateamplicons, we were able to rapidly distinguish thewild type from the mutant type at codon 43 andcodon 88 of rpsL gene due to the A to G mutation.

Validation test of HRM analysis in 30SM-resistant clinical isolates

To validate this rpsL gene mutation screeningmethod, we blindly performed the HRM on theremaining 30 SM-resistant clinical isolates (number:S1–S30) from 134 streptomycin-resistant clinicalM. tuberculosis isolates (HRM difference graph notshown). The mutation results of HRM analysis for30 SM-resistant clinical isolates were shown inTable 2. We found that 56.7% (17/30) isolates had amutation (AAG-AGG, Lys-Arg) at codon 43,13.3% (4/30) isolates had a mutation (AAG-AGG,Lys-Arg) at codon 88. In total, 70.0% (21/30) ofSM-resistant isolates harbored rpsL mutations.Furthermore, 10 strains (number: S1, S2, S3, S4,

Figure 2. HRM curve analysis of two amplicons (rpsL-a and rpsL-b). The clinical isolates were compared with H37Rvstrain by HRM graphs. It showed a clear separation between the wild and mutant isolates using a ‘‘difference graph’’.(A) Normalised HRM melt curve analysis of rpsL-a. (B) HRM difference graph of rpsL-a. (C) Normalised HRM melt curveanalysis of rpsL-b. (D) HRM difference graph of rpsL-b.

Mutation screening of rpsL gene in M. tuberculosis 125

S5, S8, S15, S18, S20, S24, in shadow) wererandomly selected from 30 clinical isolates fordirect sequencing. From the sequencing results ofthe selected 10 strains, we found that the HRMresults perfectly matched those obtained on thesame isolates by DNA sequencing.

Combined with the data from testing andvalidating examination, the sensitivity and specifi-city of HRM analysis for detecting mutations in rpsLgene at codons 43 and 88 associated with strepto-mycin resistance were 100% and 100%, respectively.

Discussion

Currently, control of MDR-TB and XDR-TB is amajor issue throughout the world. Detection ofdrug-resistant phenotypes of M. tuberculosis usingroutine methods takes several weeks. A rapid,sensitive and cost-effective method for detectingdrug-resistant phenotypes of M. tuberculosis is oneof the more urgent requirements for effectivetreatment of tuberculosis patients. An evaluationof mutation scanning by HRM analysis in this studyhas shown that HRM is a suitable and specifictechnique for mutation scanning of rpsL gene in SM-resistant clinical isolates. To our knowledge, this is

the first report of the use of HRM for analyzing rpsLgene mutations associated with SM resistance in alarge series of clinical samples.

The ability of HRM to detect these single basemutations lies in the extraordinary discriminatorypower of the advanced HRM technology. HRM isachieved by measuring the fluorescence of anamplicon as the temperature slowly increases, provid-ing greater sensitivity over traditional melt curvesbecause of the enhanced thermal resolution andability to acquire up to 1000 data points for each 1Cchange (White and Potts 2006). The PCR product size(r400 bp) for HRM are recommended. Shorteramplicons are more sensitive for detection of minorchanges and offer higher resolution (Gundry et al.2003; Liew et al. 2004). Therefore, rpsL gene wasdivided into two amplicons, rpsL-a (199 bp) and rpsL-b(235 bp) for optimizing the sensitivity of the detectionin our study. We draw a derivative plot using thestandard melt analysis software module to assesswhether there were primer dimers or non-specificproducts in the reactions. If more than one productwas found, the test was repeated or re-optimized. Ourresults showed that PCR optimization plays a crucialrole in a successful HRM analysis. By this method, pointmutations are rapidly and easily characterized,especially if gene targets possess a known SNP ascontrol.

Table 2. Summary of the results in the validation testfrom 30 isolates screened for mutations in rpsL gene.

wt, wild type; mut, mutant type; A4G, A to G mutation; w, wildtype; �, not done.

F. Wang et al.126

Furthermore, DNA sequencing and the HRMmethod were concurrently applied to investigatethe molecular mechanisms of SM resistance in M.tuberculosis clinical isolates from China. Ourresults revealed that 78.8% (82/104) of SM-resistantisolates harbored rpsL mutations. This rate is muchhigher than those of 24% in Mexico (Ramaswamyet al. 2004), 48% in Germany (Dobner et al. 1997),56–68% reported in America (Sugawara et al. 1998;Morris et al. 1995), 60% in France (Heym et al.1994). However, this rate is similar to 77.8% inJapan (Katsukawa et al. 1997). Therefore, itindicates geographic distribution of rpsL mutationsamong the SM-resistant M. tuberculosis isolatesvaries around the world. Codon 43 mutationseemed to play a major role conferring resistanceto streptomycin, showing 56.7% (59/104) isolateshad a mutation (AAG-AGG, Lys-Arg) at codon 43.Comparatively, only 22.1% (23/104) isolates had a

mutation (AAG-AGG, Lys-Arg) at codon 88conferring SM resistance. Combined mutationsinvolving codon 43 and codon 88 were not seenand had been proved to be restrictive mutations,attributing to the increased fitness cost andattenuated virulence. Only non-restrictive muta-tions such as codon 43 AAG/AGG (Lys/Arg), whichhad unaltered virulence properties, could be widelytransmitted and finally dominated in clinicalisolates (Bottger et al. 1998). Meanwhile, themutation rate at codon 43 of rpsL gene in high-resistant isolates was found to be much higher thanthose in low-resistant isolates (71.7% vs. 36.4%),whilst the mutation rate at codon 88 in high-resistant isolates was lower than those in low-resistant isolates (13.3% vs. 34.1%). Therefore, rpsLmutations associated with SM resistance in M.tuberculosis were mostly prevalent in China withcodon 43 dominating, indicating that the mutationsin rpsL gene was the important molecular mechan-ism conferring SM resistance in M. tuberculosis.

Warren et al. reported a two-step HRM protocolfor detecting the rifampin resistance-determiningregion (RRDR) in the rpoB gene in M. tuberculosiscomplex (Hoek et al. 2008). This method, named‘‘FAST-Rif’’ (‘‘fluorometric assay for susceptibilitytesting of rifampin’’), could distinguish drug-sus-ceptible and drug-resistant isolates. However, themethod did not provide information about whichnsSNP conferred resistance. In our study, we coulddetect the mutation sites of rpsL gene conferred SMresistance. We designed two amplicons of 199 bpand 235 bp, including codon 43 and codon 88,respectively. Furthermore, we used referencestrains (H37Rv, MU1, MU2) for each run, whichcould rapidly distinguish the wild type from themutant type at codon 43 and codon 88 of rpsL geneby comparing the HRM curves with the referencestrains using Rotor-Gene 6000 software.

Recently another study (Pietzka et al. 2009) hasevaluated the feasibility of mutation scanning forgenotypic drug-resistance for another antitubercu-lar drug, rifampicin. Pietzka et al. reported thatthe combined HRM analysis of all strains andisolates exhibited 95.9% sensitivity and 100%specificity in 49 MDR-TB and 19 non-MDR-TBisolates. Comparatively, we selected more clinicalisolates and performed HRM for analyzing rpsL genemutations associated with SM resistance, including134 SM-resistant clinical isolates and 20 SM-suscep-tible clinical isolates. We designed two pairs ofprimers to amplify a 199 bp amplicon (rpsL-a) and a235 bp amplicon (rpsL-b) spanning the target regionof rpsL gene, respectively. Meanwhile referencestrains (H37Rv, MU1, MU2) were used for each runso that point mutations at codons 43 and 88 were

Mutation screening of rpsL gene in M. tuberculosis 127

determined more rapidly and easily. The sensitivityand specificity of HRM analysis for detectingmutations in rpsL gene at codons 43 and 88associated with streptomycin resistance were100% and 100%, respectively, so our study furthershowed HRM analysis was an ideal screeningmethod for other drug resistance and enabledrapid, reliable, simple and cost-effective handlingof large numbers of samples.

We report here the first application of HRMtechnology for detecting mutations of rpsL geneassociated with SM resistance in M. tuberculosis.The present study showed absolute sensitivity andspecificity of the technique in clinical isolates forthe codons 43 and 88 of rpsL gene as resultsperfectly matched those obtained on the sameisolates by DNA sequencing. The HRM methodenabled rapid and high-throughput screening ofrpsL gene mutations with lower cost compared withother molecular assays, since no probes or specia-lized reagents were required. Furthermore, thesingle-tube format used in HRM would allow forhigh throughput and automation.

HRM was an extremely sensitive and specifictechnique for mutation scanning, which could beeasily integrated into clinical diagnostic pre-screening strategies. Hence, this technique canfacilitate the screening of large numbers of clinicalsamples for epidemiological studies of drug-resis-tance of M. tuberculosis, allowing for clinicians tomake decisions more rapidly for appropriate treat-ment, especially in developing countries.

Acknowledgements

The present study was supported in part by the KeyTechnologies Research and Development Program forInfectious Diseases of China (2008ZX10003003) andNational Basic Research program of China (973Program 2005CB523102). We thank Shanghai Pulmon-ary Disease Hospital for confirming phenotypic drugsusceptibility testing and providing clinical isolates ofM. tuberculosis.

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