- Email: [email protected]
Characterization of gyrA and gyrB mutations associated with fluoroquinolone resistance in Mycobacterium tuberculosis isolates from Morocco
Accepted Manuscript Title: Characterization of gyrA and gyrB mutations associated with fluoroquinolone resistance in Mycobacterium tuberculosis isolates from Morocco Authors: Imane Chaoui, Amal Oudghiri, Mohammed El Mzibri PII: DOI: Reference:
S2213-7165(17)30190-X https://doi.org/10.1016/j.jgar.2017.10.003 JGAR 513
To appear in: Received date: Revised date: Accepted date:
22-6-2017 4-10-2017 5-10-2017
Please cite this article as: Imane Chaoui, Amal Oudghiri, Mohammed El Mzibri, Characterization of gyrA and gyrB mutations associated with fluoroquinolone resistance in Mycobacterium tuberculosis isolates from Morocco (2010), https://doi.org/10.1016/j.jgar.2017.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of gyrA and gyrB mutations associated with fluoroquinolone resistance in Mycobacterium tuberculosis isolates from Morocco.
Imane Chaoui*, Amal Oudghiri, Mohammed El Mzibri.
Unité de Biologie et Recherches Médicales, Centre National de l’Energie, des Sciences et Techniques Nucléaires, BP 1382 RP. 10001, Rabat, Morocco.
*Corresponding author Dr. Imane Chaoui Unité de Biologie et Recherches Médicales, Centre National de l’Energie, des Sciences et Techniques Nucléaires, BP 1382 RP. 10001, Rabat, Morocco. Tel office: +212 537 712 03. Fax: +212 537 711 846. E-mail address: [email protected] orcid.org/0000-0002-4681-1461
Highlights
Fluoroquinolones are the cornerstone for treatment of drug-resistant TB.
About 30% of MDR isolates harbored mutations in gyrA.
All gyrA resistant strains belong to LAM Lineage raising the possible emergence of a specific clone.
The results highlight the high prevalence of FQ resistance among MDR isolates in Morocco.
Rapid detection of FQs once MDR is confirmed is critical to adjust timely the treatment.
1
Abstract Background. Fluoroquinolones (FQs) are the cornerstone for treatment of drug-resistant TB, there are the most effective second-line anti-mycobacterial drugs and are recommended for the treatment of MDR-TB. However, it’s widely accepted that FQs resistance is high among MDR-TB. Thus, characterization of mutations conferring resistance to FQs will be of a great interest for an effective and efficient management of TB resistance in Morocco. Methods: A laboratory collection of 30 MTB isolates already characterized as phenotypically and genotypically MDR and 20 pan-susceptible isolates randomly selected were enrolled in this retrospective study. The mutation profiles associated with resistance to FQs were assessed by PCR and DNA sequencing. Target sequences for two genes were examined: gyrA and gyrB. All strains had their fingerprint already established by spoligotyping. Results: Molecular analyses showed that 30% of MDR isolates harbored mutations in gyrA; the most prevalent being Ala90Thr (50%). None of the isolates harbored mutations in gyrB gene. All gyrA resistant strains belong to LAM Lineage mostly LAM9 raising the possible emergence of a specific clone (gyrA mutant/LAM9). Conclusion: The results of this preliminary study highlight the high prevalence of FQs resistance among MDR isolates in Morocco and consequently the need for rapid detection of FQs once MDR is confirmed to adjust timely the treatment and to interrupt the propagation of more severe form of MTB drug resistance.
Keywords: Morocco, Mycobacterium tuberculosis, Fluoroquinolones resistance, gyrA, gyrB, sequencing.
Introduction During last decades, the emergence of drug resistance has made an effective control strategy for tuberculosis (TB) indispensable. Tuberculosis is hampered by the rapid spread of multidrug /extensively- drug-resistant TB (MDR/XDR-TB), both in new and in previously treated cases, which warrants the need for decisive action to adjust therapies and prevent further resistance (1). Fluoroquinolones (FQs) are the most effective antimicrobial agents used to treat MDR-TB. Unfortunately, FQs have been widely prescribed in the treatment of undiagnosed respiratory bacterial infections (2), hence; their uncontrolled and inappropriate use may contribute to FQs resistance in M. tuberculosis (MTB), which may influence the clinical outcome for MDR-TB
2
patients. Thus, recognizing the sensitive/resistant FQs status will provide new insights to develop the appropriate regimen for MDR patients. Resistance to FQs, such as Ofloxacin (OFX), commonly used to treat MDR-TB is thought to be mediated by mutations in the target genes: gyrA and, less frequently, gyrB, which encode the respective subunits of the DNA topoisomerase gyrase (3). Most mutations conferring resistance to FQs are known to be accumulated in two short discrete regions of gyrA and gyrB genes termed the Quinolone Resistance-Determining Regions (QRDRs) (4, 5). FQ resistance is increasingly reported worldwide. Hence, the present preliminary study was planned to assess the mutational status of gyrA and gyrB genes on both pan-susceptible and MDR-TB isolates from Morocco and to analyze the relationship between genotypes and FQs drug resistance patterns.
Materiel and methods Sample collection A collection of 30 MDR and 20 pan-susceptible strains selected from our laboratory collection was enrolled in this work (6, 7). These strains were isolated from patients with pulmonary tuberculosis. Clinical status of patients revealed that 36% (18/50) were new cases, whereas 30% (15/50) were relapsed, 20% (10/50) failed to treatment and 14% defaulted (7/50). It should be underlined that: (i) only good quality bacterial lysis was subject to the present study: in fact, many samples were damaged because of several cycles of freezing-thawing,(ii) molecular characterization of mutations conferring resistance to RIF and INH was already realized; (iii) molecular typing of clinical isolates by Spoligotyping was already performed (8). The characterization of genetic mutations associated with FQs resistance (gyrA and gyrB) was performed by PCR amplification and DNA sequencing.
PCR amplification of gyrA and gyrB genes The gyrA and gyrB genes were amplified by PCR using the corresponding primers (Feuerriegel et al, 2009). Amplification reactions were performed in a total volume of 50 μL containing 0.5 mM of each primer, 2.5 mM of each dNTP, 25 mM MgCl2, 1 unit of Hotstar Taq DNA polymerase (Invitrogen, Saint Aubin, France), and 2μL of crude DNA (bacterial lysis) in 1 × Taq polymerase buffer. The mixtures were first denatured at 94°C for 7 minutes, 3
then thirty-five cycles of PCR were then performed, with denaturation at 94°C for one minute, primer annealing for one minute at the corresponding melting temperature, and primer extension for one minute at 72°C. At the end of the last cycle, the mixture was incubated at 72°C for 7 minutes. For each reaction, a negative control containing H2O in which DNA template was omitted from the amplification mixture and a positive control containing DNA from H37Rv strain were included.
DNA sequencing Direct sequencing of amplicons was done using Big Dye Terminator kit version 3.1 on an ABI 3130XL DNA analyzer (Applied Biosystems, Foster city, CA, USA) as described previously (6, 9). For each amplicon, both strands were sequenced, in independent reactions. The resulting electrophoregrams were analyzed by Mega V software.
Results Both susceptible (n=20) and MDR isolates (n=30) were subject to DNA sequencing for gyrA and gyrB genes. None of susceptible isolates harbored any SNP in the respective genes. Among the genotypically MDR strains, 9 (30%) had mutations in gyrA gene but none of them harbored any SNP in gyrB gene. Genotypic results regarding resistance to FQs are reported in Table I. Our results showed that the most recorded mutation in gyrA gene is the substitution of GCG>ACG at codon 90 (Ala90Thr) accounting for 55.6% of all cases. Other mutations at codon 94; Asp94Ala (GAC>GCC); Asp94His (GAC>CAC) and Asp94Val (GAC>GTC), were found respectively in 2, 1 and 1 isolates. Moreover, the substitution Thr95Ser (ACC>AGC) in gyrA gene was found in 4 isolates. The silent mutation TCG (Ser)>TCA (Ser) at codon 91 was identified in 7 isolates; these SNPs are known to be not associated to any resistance (Table II). Of note, the combination of sensitive/resistant profile and clinical status of the respective patients showed that almost sensitive strains were new cases whereas MDR strains were mainly isolated from patients who were relapsed, failed to treatment or defaulted. Of note, all strains harboring mutations in gyrA gene were isolated from patients with previous TB history. In fact, among the 9 pre-XDR strains, 5 were isolated from patients with failure, 3 from patients with relapse and 1 case from a patient with treatment default. Of particular interest, when combining strain typing results with genotypic drug resistance data, it seems that all isolates carrying mutation in gyrA gene, carried also mutations in rpoB 4
and katG genes and belong to Latin-American-Mediterranean lineage namely LAM4 and LAM9 (Table III).
Discussion Early diagnosis of MDR/XDR-TB is crucially important for rational regimen to prevent further transmission of drug resistant TB. In the present study, we focused our interest on FQs resistance. Our results showed that 30% of MDR-TB isolates carried mutations in gyrA gene at codons 90 (55.6%) and 94 (44.4%); Ala90Thr being the most common one. Our findings are in agreement with previously reported data, even though it was suggested that mutations at codon 94, rather than codon 90, are favored globally (10 - 12). Three different substitutions were observed at position 94 corroborating the findings of previous studies (13). These mutations have been previously described as mediating resistance to FQs (4, 5). Mutations in gyrB had not been found in this collection. Worldwide, mutations in gyrB gene occur at low frequency, even though mutations in gyrB should also be considered when screening for FQs resistant strains (9, 14). Of particular interest, all gyrA mutants displayed mutations in rpoB gene at codon 531 and katG gene at codon 315, suggesting that MDR isolates with such SNPs are more likely to develop FQs resistant through the acquisition of gyrA mutations. Although not related to resistance, four isolates had the ACC>AGC mutation at codon 95 of gyrA, indicating that these isolates belonged to the Principal Genetic Group 1 or 2 of the M. tuberculosis complex. Our results implies that the MDR MTB isolates are already resistant to FQs which may compromise the response to treatment, moreover the high prevalence of FQs resistance based on gyrA mutations could be correlated with the patient’s previous exposure to FQs (15). Of note, none of pan-susceptible isolates displayed mutations neither in gyrA nor in gyrB genes indicating that the patients have not been exposed previously to FQs. The correlation between genotypic and phenotypic data could not be performed because DST results for FQs were not available. Indeed, at the study period (2014), no laboratory in Morocco had second-line DST capability. The combination of spoligotyping patterns and gyrA mutations, associated with FQ resistance) results showed a marked predominance of one single strain family (LAM) within FQs resistant isolates, this finding deserves more investigations. In fact, an association of certain strain families with Pre X/XDR-TB could be attributed to their relatively more effective transmission as MDR strains or, alternatively, may be explained by an enhanced intrinsic capacity to acquire resistance to second-line anti-TB drugs (16); further studies on 5
large number of resistant isolates are warranted to provide a more accurate picture of the epidemiology of drug resistant MTB strains in Morocco and to determine the rate of recent transmission among the population. Of note, none of MDR strains belongs to Beijing clade, widely reported to have significant associations with drug-resistance and assumed to be responsible for the emergence and spread of MDR-TB. Worldwide, the emergence of XDR MTB strains led to the increased use of second line drugs (SLDs) for TB treatment and subsequently to the need to extend DST to SLDs. Unlikely; there are still methodological problems with current DST for SLDs. Moreover, DST for SLDs is not performed in low income countries where MDR-TB prevalence is high and MDR-TB treatment is not yet individualized (17). There is a current need for the development and implementation of rapid molecular tests that detect mutations associated with MDR as well as second line drug resistance. Conventional culture-based techniques are technically demanding and have long turnaround times. Molecular techniques, such as DNA sequencing method presented here, provide specific and prompt drug resistance data that may guide treatment regimens. However, the development of genetic assays requires the verification of molecular data (for instance, mutations associated with FQs) with respect to conventional drug susceptibility results (18).
Conclusion The results of this study demonstrate the utility of detection of mutations associated with resistance to FQs which is crucial for predicting phenotypic resistance to FQs, for optimizing TB treatment and preventing further transmission of drug resistant MTB strains. These findings emphasize the need for implementation of DST to SLD in routine along with accurate and rapid molecular tests for the detection of FQ-resistant mutations once MDR-TB is diagnosed.
Declarations Funding: This study was funded in part by the International Atomic Energy Agency under the RAF6040 project and Regional Office for the Eastern Mediterranean/World Health organization under the RPC/RAB&GH 10/11-03 project.
6
Ethical Approval: Not required
Competing Interests: No
7
References 1.World Health Organization. 2013. Multidrug and extensively drug-resistant TB (M/XDRTB): Global tuberculosis report. World Health Organization, Geneva, Switzerland. Available at http://apps.who.int/iris/bitstream/10665/91355/1/9789241564656_eng.pdf. Assessed on June 16th, 2017. 2. Zhang Z, Lu J, Wang Y, Pang Y, Zhao Y. Prevalence and molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis isolates in China. Antimicrob Agents Chemother 2014;58:364-9. 3. Mayer C, Takiff H. The Molecular Genetics of Fluoroquinolone Resistance in Mycobacterium tuberculosis. Microbiol Spectr 2014; 2:MGM2-0009-2013. 4. Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis. Tuber Lung Dis 1998;79:3-29. 5. Sandgren A, StrongM, Muthukrishnan P, WeinerBK, Church GM, MurrayMB. Tuberculosis Drug Resistance Mutation Database. PLoS Med 6(2):e1000002. 6. Zakham F, Chaoui I, Echchaoui AH, Chetioui F, Elmessaoudi MD, Ennaji MM, Abid M, El Mzibri M. Direct sequencing for rapid detection of multidrug resistant Mycobacterium tuberculosis strains in Morocco. Infect Drug Resist 2013;28:207-13. 7.Chaoui I, Atalhi I, Sabouni R, Akrim M, Abid M, Amzazi S, El Mzibri M. A rifoligotyping assay: an alternative method for rapid detection of rifampicin resistance in Mycobacterium tuberculosis isolates from Morocco. Biotechnol and Biotechnol Equip 2014;28:1095-102 8.Chaoui I, Zozio T, Lahlou O, Sabouni R, Abid M, Elaouad R, Akrim M, Amzazi S, Rastogi N, El Mzibri M. Contribution of spoligotyping and MIRU-VNTRs to characterize prevalent Mycobacterium tuberculosis genotypes infecting tuberculosis patients in Morocco. Infect Genet Evol 2014;S1567-1348. 9. Feuerriegel S, Cox HS, Zarkua N, Karimovich HA, Braker K, Rüsch-Gerdes S, Niemann S. Sequence analyses of just four genes to detect extensively drug-resistant Mycobacterium tuberculosis strains in multidrug-resistant tuberculosis patients undergoing treatment. Antimicrob. Agents. Chemother 2009;53:3353-6. 10. Hoshide M, Qian L, Rodrigues C, Warren R, Victor T, Evasco HB, Tupasi T, Crudu V, Douglas JT. Geographical differences associated with single-nucleotide polymorphisms (SNPs) in nine gene targets among resistant clinical isolates of Mycobacterium tuberculosis. J Clin Microbiol 2014;52(5):1322-9. 11. Avalos E, Catanzaro D, Catanzaro A, Ganiats T, Brodine S, Alcaraz J, Rodwell T. Frequency and geographic distribution of gyrA and gyrB mutations associated with 8
fluoroquinolone resistance in clinical Mycobacterium tuberculosis isolates: a systematic review. PLoSOne 2015;10(3):e0120470. 12. Chen J, Peng P, Du Y, Ren Y, Chen L, Rao Y, Wang W. Early detection of multidrugand pre-extensively drug-resistant tuberculosis from smear positive sputum by direct sequencing. BMC Infect Dis 2017;17:300. 13. Devasia R, Blackman A, Eden S, Li H, Maruri F, Shintani A, Alexander C, Kaiga A, Stratton CW, Warkentin J, Tang YW, Sterling TR. High proportion of fluoroquinoloneresistant Mycobacterium tuberculosis isolates with novel gyrase polymorphisms and a gyrA region associated with fluoroquinolone susceptibility. J Clin Microbiol 2012;50(4):1390-6. 14. Juarez-Eusebio DM, Munro-Rojas D, Muñiz-Salazar R, Laniado-Laborín R, MartinezGuarneros JA, Flores-López CA, Zenteno-Cuevas R. Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates from high prevalence tuberculosis states in Mexico. Infect Genet Evol 2016; S1567-1348(16)30395-1 15. Wang JY, Lee LN, Lai HC, Wang SK, Jan IS, Yu CJ, Hsueh PR, Yang PC. Fluoroquinolone resistance in Mycobacterium tuberculosis isolates: associated genetic mutations and relationship to antimicrobial exposure. J Antimicrob Chemother 2007;59(5):860-5. 16. Chihota VN, Müller B, Mlambo CK, Pillay M, Tait M, Streicher EM, Marais E, van der Spuy GD, Hanekom M, Coetzee G, Trollip A, Hayes C, Bosman ME, Gey van Pittius NC, Victor TC, van Helden PD, Warren RB. Population structure of multi- and extensively drugresistant Mycobacterium tuberculosis strains in South Africa. J Clin Microbiol 2012;50(3):995-1002. 17. Surcouf C, Heng S, Pierre-Audigier C, Cadet-Daniel V, Namouchi A, Murray A, et al. Molecular detection of fluoroquinolone-resistance in multi-drug resistant tuberculosis in Combodia suggests low association with XDR phenotypes. BMC Infect Dis 2011;11:255. 18. Campbell PJ, Morlock GP, Sikes RD, Dalton TL, Metchock B, Starks AM, et al. Molecular Detection of Mutations Associated with First- and Second-Line Drug Resistance Compared with Conventional Drug Susceptibility Testing of Mycobacterium tuberculosis. Antimicrob Agents and Chemother 2011;55:2032-41.
9
Table I. Details results of mutations conferring resistance to FQs Gene
gyrA
Position
Nucleotide change
Frequency
90
GCG→ACG Ala>Thr
5
94
GAC→GCC Asp >Ala
2
94
GAC→CAC Asp >His
1
94
GAC→GTC Asp >Val
1
Total
9
Table II. Details results of SNPs not involved in MTB resistance to FQs Gene
Position
Nucleotide change
Frequency
gyrA
91
TCG→TCA Ser>Ser
7
95
ACC→AGC Thr>Ser
4
10
Total 11
Table III. Details results of DST, sequencing and spoligotyping of 20 pan-Susceptible and 30 MDR MTB isolates.
Multidrug reistant
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Pan-Susceptible
ID DST
rpoB wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt TCG531TTG TCG531TGG TCG531TTG TCG531TTG TCG531TTG TCG531TTG TCG531TTG TCG531TTG CAC526TAC TCG531TTG GAC516GTC TCG531TTG TCG531TGG TCG531TTG CAC526TAC TCG531TTG TCG531TGG TCG531TTG TCG531TTG GAC516GTC TCG531TTG TCG531TTG TCG531TTG TCG531TTG TCG531TTG CAC526TAC TCG531TTG TCG531TTG TCG531TTG
Sequencing katG wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC
Spoligotype description (binary)
gyrA
wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt GCG90ACG GCG90ACG GCG90ACG GCG90ACG; ACC95AGC GAC94GCC GAC94GCC GAC94CAC; TCG91TCA GAC94GTC wt wt TCG91TCA ACC95AGC wt wt TCG91TCA ACC95AGC wt TCG91TCA wt wt TCG91TCA wt ACC95AGC TCG91TCA wt TCG91TCA wt wt GCG90ACG
SIT: Shared International Type
11
SIT
Lineage
1058 1074 2338 2576 2898 3 33 36 36 42 42 42 42 42 47 47 50 50 50 53 60 509 33 1075 1064 1064 42 60 47 53 2331 53 42 42 orphan 71 177 93 1068 53 42 53 60 509 53 34 53 53 1075
T2 LAM9 H3 LAM5 T1 H3 LAM3 H3 H3 LAM9 LAM9 LAM9 LAM9 LAM9 H1 H1 H3 H3 H3 T1 LAM4 LAM9 LAM3 LAM9 LAM9 LAM9 LAM9 LAM4 H1 T1 LAM9 T1 LAM9 LAM9 T1 S LAM9 LAM5 S T1 LAM9 T1 LAM4 LAM9 T1 S T1 T1 LAM9
S2213-7165(17)30190-X https://doi.org/10.1016/j.jgar.2017.10.003 JGAR 513
To appear in: Received date: Revised date: Accepted date:
22-6-2017 4-10-2017 5-10-2017
Please cite this article as: Imane Chaoui, Amal Oudghiri, Mohammed El Mzibri, Characterization of gyrA and gyrB mutations associated with fluoroquinolone resistance in Mycobacterium tuberculosis isolates from Morocco (2010), https://doi.org/10.1016/j.jgar.2017.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of gyrA and gyrB mutations associated with fluoroquinolone resistance in Mycobacterium tuberculosis isolates from Morocco.
Imane Chaoui*, Amal Oudghiri, Mohammed El Mzibri.
Unité de Biologie et Recherches Médicales, Centre National de l’Energie, des Sciences et Techniques Nucléaires, BP 1382 RP. 10001, Rabat, Morocco.
*Corresponding author Dr. Imane Chaoui Unité de Biologie et Recherches Médicales, Centre National de l’Energie, des Sciences et Techniques Nucléaires, BP 1382 RP. 10001, Rabat, Morocco. Tel office: +212 537 712 03. Fax: +212 537 711 846. E-mail address: [email protected] orcid.org/0000-0002-4681-1461
Highlights
Fluoroquinolones are the cornerstone for treatment of drug-resistant TB.
About 30% of MDR isolates harbored mutations in gyrA.
All gyrA resistant strains belong to LAM Lineage raising the possible emergence of a specific clone.
The results highlight the high prevalence of FQ resistance among MDR isolates in Morocco.
Rapid detection of FQs once MDR is confirmed is critical to adjust timely the treatment.
1
Abstract Background. Fluoroquinolones (FQs) are the cornerstone for treatment of drug-resistant TB, there are the most effective second-line anti-mycobacterial drugs and are recommended for the treatment of MDR-TB. However, it’s widely accepted that FQs resistance is high among MDR-TB. Thus, characterization of mutations conferring resistance to FQs will be of a great interest for an effective and efficient management of TB resistance in Morocco. Methods: A laboratory collection of 30 MTB isolates already characterized as phenotypically and genotypically MDR and 20 pan-susceptible isolates randomly selected were enrolled in this retrospective study. The mutation profiles associated with resistance to FQs were assessed by PCR and DNA sequencing. Target sequences for two genes were examined: gyrA and gyrB. All strains had their fingerprint already established by spoligotyping. Results: Molecular analyses showed that 30% of MDR isolates harbored mutations in gyrA; the most prevalent being Ala90Thr (50%). None of the isolates harbored mutations in gyrB gene. All gyrA resistant strains belong to LAM Lineage mostly LAM9 raising the possible emergence of a specific clone (gyrA mutant/LAM9). Conclusion: The results of this preliminary study highlight the high prevalence of FQs resistance among MDR isolates in Morocco and consequently the need for rapid detection of FQs once MDR is confirmed to adjust timely the treatment and to interrupt the propagation of more severe form of MTB drug resistance.
Keywords: Morocco, Mycobacterium tuberculosis, Fluoroquinolones resistance, gyrA, gyrB, sequencing.
Introduction During last decades, the emergence of drug resistance has made an effective control strategy for tuberculosis (TB) indispensable. Tuberculosis is hampered by the rapid spread of multidrug /extensively- drug-resistant TB (MDR/XDR-TB), both in new and in previously treated cases, which warrants the need for decisive action to adjust therapies and prevent further resistance (1). Fluoroquinolones (FQs) are the most effective antimicrobial agents used to treat MDR-TB. Unfortunately, FQs have been widely prescribed in the treatment of undiagnosed respiratory bacterial infections (2), hence; their uncontrolled and inappropriate use may contribute to FQs resistance in M. tuberculosis (MTB), which may influence the clinical outcome for MDR-TB
2
patients. Thus, recognizing the sensitive/resistant FQs status will provide new insights to develop the appropriate regimen for MDR patients. Resistance to FQs, such as Ofloxacin (OFX), commonly used to treat MDR-TB is thought to be mediated by mutations in the target genes: gyrA and, less frequently, gyrB, which encode the respective subunits of the DNA topoisomerase gyrase (3). Most mutations conferring resistance to FQs are known to be accumulated in two short discrete regions of gyrA and gyrB genes termed the Quinolone Resistance-Determining Regions (QRDRs) (4, 5). FQ resistance is increasingly reported worldwide. Hence, the present preliminary study was planned to assess the mutational status of gyrA and gyrB genes on both pan-susceptible and MDR-TB isolates from Morocco and to analyze the relationship between genotypes and FQs drug resistance patterns.
Materiel and methods Sample collection A collection of 30 MDR and 20 pan-susceptible strains selected from our laboratory collection was enrolled in this work (6, 7). These strains were isolated from patients with pulmonary tuberculosis. Clinical status of patients revealed that 36% (18/50) were new cases, whereas 30% (15/50) were relapsed, 20% (10/50) failed to treatment and 14% defaulted (7/50). It should be underlined that: (i) only good quality bacterial lysis was subject to the present study: in fact, many samples were damaged because of several cycles of freezing-thawing,(ii) molecular characterization of mutations conferring resistance to RIF and INH was already realized; (iii) molecular typing of clinical isolates by Spoligotyping was already performed (8). The characterization of genetic mutations associated with FQs resistance (gyrA and gyrB) was performed by PCR amplification and DNA sequencing.
PCR amplification of gyrA and gyrB genes The gyrA and gyrB genes were amplified by PCR using the corresponding primers (Feuerriegel et al, 2009). Amplification reactions were performed in a total volume of 50 μL containing 0.5 mM of each primer, 2.5 mM of each dNTP, 25 mM MgCl2, 1 unit of Hotstar Taq DNA polymerase (Invitrogen, Saint Aubin, France), and 2μL of crude DNA (bacterial lysis) in 1 × Taq polymerase buffer. The mixtures were first denatured at 94°C for 7 minutes, 3
then thirty-five cycles of PCR were then performed, with denaturation at 94°C for one minute, primer annealing for one minute at the corresponding melting temperature, and primer extension for one minute at 72°C. At the end of the last cycle, the mixture was incubated at 72°C for 7 minutes. For each reaction, a negative control containing H2O in which DNA template was omitted from the amplification mixture and a positive control containing DNA from H37Rv strain were included.
DNA sequencing Direct sequencing of amplicons was done using Big Dye Terminator kit version 3.1 on an ABI 3130XL DNA analyzer (Applied Biosystems, Foster city, CA, USA) as described previously (6, 9). For each amplicon, both strands were sequenced, in independent reactions. The resulting electrophoregrams were analyzed by Mega V software.
Results Both susceptible (n=20) and MDR isolates (n=30) were subject to DNA sequencing for gyrA and gyrB genes. None of susceptible isolates harbored any SNP in the respective genes. Among the genotypically MDR strains, 9 (30%) had mutations in gyrA gene but none of them harbored any SNP in gyrB gene. Genotypic results regarding resistance to FQs are reported in Table I. Our results showed that the most recorded mutation in gyrA gene is the substitution of GCG>ACG at codon 90 (Ala90Thr) accounting for 55.6% of all cases. Other mutations at codon 94; Asp94Ala (GAC>GCC); Asp94His (GAC>CAC) and Asp94Val (GAC>GTC), were found respectively in 2, 1 and 1 isolates. Moreover, the substitution Thr95Ser (ACC>AGC) in gyrA gene was found in 4 isolates. The silent mutation TCG (Ser)>TCA (Ser) at codon 91 was identified in 7 isolates; these SNPs are known to be not associated to any resistance (Table II). Of note, the combination of sensitive/resistant profile and clinical status of the respective patients showed that almost sensitive strains were new cases whereas MDR strains were mainly isolated from patients who were relapsed, failed to treatment or defaulted. Of note, all strains harboring mutations in gyrA gene were isolated from patients with previous TB history. In fact, among the 9 pre-XDR strains, 5 were isolated from patients with failure, 3 from patients with relapse and 1 case from a patient with treatment default. Of particular interest, when combining strain typing results with genotypic drug resistance data, it seems that all isolates carrying mutation in gyrA gene, carried also mutations in rpoB 4
and katG genes and belong to Latin-American-Mediterranean lineage namely LAM4 and LAM9 (Table III).
Discussion Early diagnosis of MDR/XDR-TB is crucially important for rational regimen to prevent further transmission of drug resistant TB. In the present study, we focused our interest on FQs resistance. Our results showed that 30% of MDR-TB isolates carried mutations in gyrA gene at codons 90 (55.6%) and 94 (44.4%); Ala90Thr being the most common one. Our findings are in agreement with previously reported data, even though it was suggested that mutations at codon 94, rather than codon 90, are favored globally (10 - 12). Three different substitutions were observed at position 94 corroborating the findings of previous studies (13). These mutations have been previously described as mediating resistance to FQs (4, 5). Mutations in gyrB had not been found in this collection. Worldwide, mutations in gyrB gene occur at low frequency, even though mutations in gyrB should also be considered when screening for FQs resistant strains (9, 14). Of particular interest, all gyrA mutants displayed mutations in rpoB gene at codon 531 and katG gene at codon 315, suggesting that MDR isolates with such SNPs are more likely to develop FQs resistant through the acquisition of gyrA mutations. Although not related to resistance, four isolates had the ACC>AGC mutation at codon 95 of gyrA, indicating that these isolates belonged to the Principal Genetic Group 1 or 2 of the M. tuberculosis complex. Our results implies that the MDR MTB isolates are already resistant to FQs which may compromise the response to treatment, moreover the high prevalence of FQs resistance based on gyrA mutations could be correlated with the patient’s previous exposure to FQs (15). Of note, none of pan-susceptible isolates displayed mutations neither in gyrA nor in gyrB genes indicating that the patients have not been exposed previously to FQs. The correlation between genotypic and phenotypic data could not be performed because DST results for FQs were not available. Indeed, at the study period (2014), no laboratory in Morocco had second-line DST capability. The combination of spoligotyping patterns and gyrA mutations, associated with FQ resistance) results showed a marked predominance of one single strain family (LAM) within FQs resistant isolates, this finding deserves more investigations. In fact, an association of certain strain families with Pre X/XDR-TB could be attributed to their relatively more effective transmission as MDR strains or, alternatively, may be explained by an enhanced intrinsic capacity to acquire resistance to second-line anti-TB drugs (16); further studies on 5
large number of resistant isolates are warranted to provide a more accurate picture of the epidemiology of drug resistant MTB strains in Morocco and to determine the rate of recent transmission among the population. Of note, none of MDR strains belongs to Beijing clade, widely reported to have significant associations with drug-resistance and assumed to be responsible for the emergence and spread of MDR-TB. Worldwide, the emergence of XDR MTB strains led to the increased use of second line drugs (SLDs) for TB treatment and subsequently to the need to extend DST to SLDs. Unlikely; there are still methodological problems with current DST for SLDs. Moreover, DST for SLDs is not performed in low income countries where MDR-TB prevalence is high and MDR-TB treatment is not yet individualized (17). There is a current need for the development and implementation of rapid molecular tests that detect mutations associated with MDR as well as second line drug resistance. Conventional culture-based techniques are technically demanding and have long turnaround times. Molecular techniques, such as DNA sequencing method presented here, provide specific and prompt drug resistance data that may guide treatment regimens. However, the development of genetic assays requires the verification of molecular data (for instance, mutations associated with FQs) with respect to conventional drug susceptibility results (18).
Conclusion The results of this study demonstrate the utility of detection of mutations associated with resistance to FQs which is crucial for predicting phenotypic resistance to FQs, for optimizing TB treatment and preventing further transmission of drug resistant MTB strains. These findings emphasize the need for implementation of DST to SLD in routine along with accurate and rapid molecular tests for the detection of FQ-resistant mutations once MDR-TB is diagnosed.
Declarations Funding: This study was funded in part by the International Atomic Energy Agency under the RAF6040 project and Regional Office for the Eastern Mediterranean/World Health organization under the RPC/RAB&GH 10/11-03 project.
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Ethical Approval: Not required
Competing Interests: No
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References 1.World Health Organization. 2013. Multidrug and extensively drug-resistant TB (M/XDRTB): Global tuberculosis report. World Health Organization, Geneva, Switzerland. Available at http://apps.who.int/iris/bitstream/10665/91355/1/9789241564656_eng.pdf. Assessed on June 16th, 2017. 2. Zhang Z, Lu J, Wang Y, Pang Y, Zhao Y. Prevalence and molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis isolates in China. Antimicrob Agents Chemother 2014;58:364-9. 3. Mayer C, Takiff H. The Molecular Genetics of Fluoroquinolone Resistance in Mycobacterium tuberculosis. Microbiol Spectr 2014; 2:MGM2-0009-2013. 4. Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis. Tuber Lung Dis 1998;79:3-29. 5. Sandgren A, StrongM, Muthukrishnan P, WeinerBK, Church GM, MurrayMB. Tuberculosis Drug Resistance Mutation Database. PLoS Med 6(2):e1000002. 6. Zakham F, Chaoui I, Echchaoui AH, Chetioui F, Elmessaoudi MD, Ennaji MM, Abid M, El Mzibri M. Direct sequencing for rapid detection of multidrug resistant Mycobacterium tuberculosis strains in Morocco. Infect Drug Resist 2013;28:207-13. 7.Chaoui I, Atalhi I, Sabouni R, Akrim M, Abid M, Amzazi S, El Mzibri M. A rifoligotyping assay: an alternative method for rapid detection of rifampicin resistance in Mycobacterium tuberculosis isolates from Morocco. Biotechnol and Biotechnol Equip 2014;28:1095-102 8.Chaoui I, Zozio T, Lahlou O, Sabouni R, Abid M, Elaouad R, Akrim M, Amzazi S, Rastogi N, El Mzibri M. Contribution of spoligotyping and MIRU-VNTRs to characterize prevalent Mycobacterium tuberculosis genotypes infecting tuberculosis patients in Morocco. Infect Genet Evol 2014;S1567-1348. 9. Feuerriegel S, Cox HS, Zarkua N, Karimovich HA, Braker K, Rüsch-Gerdes S, Niemann S. Sequence analyses of just four genes to detect extensively drug-resistant Mycobacterium tuberculosis strains in multidrug-resistant tuberculosis patients undergoing treatment. Antimicrob. Agents. Chemother 2009;53:3353-6. 10. Hoshide M, Qian L, Rodrigues C, Warren R, Victor T, Evasco HB, Tupasi T, Crudu V, Douglas JT. Geographical differences associated with single-nucleotide polymorphisms (SNPs) in nine gene targets among resistant clinical isolates of Mycobacterium tuberculosis. J Clin Microbiol 2014;52(5):1322-9. 11. Avalos E, Catanzaro D, Catanzaro A, Ganiats T, Brodine S, Alcaraz J, Rodwell T. Frequency and geographic distribution of gyrA and gyrB mutations associated with 8
fluoroquinolone resistance in clinical Mycobacterium tuberculosis isolates: a systematic review. PLoSOne 2015;10(3):e0120470. 12. Chen J, Peng P, Du Y, Ren Y, Chen L, Rao Y, Wang W. Early detection of multidrugand pre-extensively drug-resistant tuberculosis from smear positive sputum by direct sequencing. BMC Infect Dis 2017;17:300. 13. Devasia R, Blackman A, Eden S, Li H, Maruri F, Shintani A, Alexander C, Kaiga A, Stratton CW, Warkentin J, Tang YW, Sterling TR. High proportion of fluoroquinoloneresistant Mycobacterium tuberculosis isolates with novel gyrase polymorphisms and a gyrA region associated with fluoroquinolone susceptibility. J Clin Microbiol 2012;50(4):1390-6. 14. Juarez-Eusebio DM, Munro-Rojas D, Muñiz-Salazar R, Laniado-Laborín R, MartinezGuarneros JA, Flores-López CA, Zenteno-Cuevas R. Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates from high prevalence tuberculosis states in Mexico. Infect Genet Evol 2016; S1567-1348(16)30395-1 15. Wang JY, Lee LN, Lai HC, Wang SK, Jan IS, Yu CJ, Hsueh PR, Yang PC. Fluoroquinolone resistance in Mycobacterium tuberculosis isolates: associated genetic mutations and relationship to antimicrobial exposure. J Antimicrob Chemother 2007;59(5):860-5. 16. Chihota VN, Müller B, Mlambo CK, Pillay M, Tait M, Streicher EM, Marais E, van der Spuy GD, Hanekom M, Coetzee G, Trollip A, Hayes C, Bosman ME, Gey van Pittius NC, Victor TC, van Helden PD, Warren RB. Population structure of multi- and extensively drugresistant Mycobacterium tuberculosis strains in South Africa. J Clin Microbiol 2012;50(3):995-1002. 17. Surcouf C, Heng S, Pierre-Audigier C, Cadet-Daniel V, Namouchi A, Murray A, et al. Molecular detection of fluoroquinolone-resistance in multi-drug resistant tuberculosis in Combodia suggests low association with XDR phenotypes. BMC Infect Dis 2011;11:255. 18. Campbell PJ, Morlock GP, Sikes RD, Dalton TL, Metchock B, Starks AM, et al. Molecular Detection of Mutations Associated with First- and Second-Line Drug Resistance Compared with Conventional Drug Susceptibility Testing of Mycobacterium tuberculosis. Antimicrob Agents and Chemother 2011;55:2032-41.
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Table I. Details results of mutations conferring resistance to FQs Gene
gyrA
Position
Nucleotide change
Frequency
90
GCG→ACG Ala>Thr
5
94
GAC→GCC Asp >Ala
2
94
GAC→CAC Asp >His
1
94
GAC→GTC Asp >Val
1
Total
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Table II. Details results of SNPs not involved in MTB resistance to FQs Gene
Position
Nucleotide change
Frequency
gyrA
91
TCG→TCA Ser>Ser
7
95
ACC→AGC Thr>Ser
4
10
Total 11
Table III. Details results of DST, sequencing and spoligotyping of 20 pan-Susceptible and 30 MDR MTB isolates.
Multidrug reistant
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
Pan-Susceptible
ID DST
rpoB wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt TCG531TTG TCG531TGG TCG531TTG TCG531TTG TCG531TTG TCG531TTG TCG531TTG TCG531TTG CAC526TAC TCG531TTG GAC516GTC TCG531TTG TCG531TGG TCG531TTG CAC526TAC TCG531TTG TCG531TGG TCG531TTG TCG531TTG GAC516GTC TCG531TTG TCG531TTG TCG531TTG TCG531TTG TCG531TTG CAC526TAC TCG531TTG TCG531TTG TCG531TTG
Sequencing katG wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC AGC315ACC
Spoligotype description (binary)
gyrA
wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt wt GCG90ACG GCG90ACG GCG90ACG GCG90ACG; ACC95AGC GAC94GCC GAC94GCC GAC94CAC; TCG91TCA GAC94GTC wt wt TCG91TCA ACC95AGC wt wt TCG91TCA ACC95AGC wt TCG91TCA wt wt TCG91TCA wt ACC95AGC TCG91TCA wt TCG91TCA wt wt GCG90ACG
SIT: Shared International Type
11
SIT
Lineage
1058 1074 2338 2576 2898 3 33 36 36 42 42 42 42 42 47 47 50 50 50 53 60 509 33 1075 1064 1064 42 60 47 53 2331 53 42 42 orphan 71 177 93 1068 53 42 53 60 509 53 34 53 53 1075
T2 LAM9 H3 LAM5 T1 H3 LAM3 H3 H3 LAM9 LAM9 LAM9 LAM9 LAM9 H1 H1 H3 H3 H3 T1 LAM4 LAM9 LAM3 LAM9 LAM9 LAM9 LAM9 LAM4 H1 T1 LAM9 T1 LAM9 LAM9 T1 S LAM9 LAM5 S T1 LAM9 T1 LAM4 LAM9 T1 S T1 T1 LAM9