Multiplex PCR Amplimer Conformation Analysis for Rapid Detection of gyrA Mutations in Fluoroquinolone-Resistant Mycobacterium tuberculosis Clinical Isolates

Abstract

A new strategy known as multiplex PCR amplimer conformation was developed for detection of mutation in the gyrA gene of 138 clinical isolates of Mycobacterium tuberculosis. The method generated a single-stranded and heteroduplex DNA banding pattern of multiplex PCR amplimers of the region of interest that was extremely sensitive to specific mutations, thus enabling much more sensitive and reliable mutation analysis compared to the standard single-stranded conformation polymorphism technique. The genetic profiles of the gyrA gene of the 138 isolates as detected by MPAC were confirmed by nucleotide sequencing and were found to correlate strongly with the in vitro susceptibilities of the mutant strains to six fluoroquinolones (ofloxacin, levofloxacin, sparfloxacin, moxifloxacin, gatifloxacin, and sitafloxacin). All 32 isolates that contained gyrA mutations exhibited cross-resistance to the six fluoroquinolones (ofloxacin MIC for 90% of strains > 16 mg/liter), although moxifloxacin, gatifloxacin, and sitafloxacin (MIC for 90% of strains < 4 mg/liter) were apparently more active than ofloxacin, levofloxacin, and sparfloxacin (MIC for 90% of strains > 16 mg/liter). All gyrA mutations were clustered in codons 90, 91, and 94, and aspartic acid 94 was most frequently mutated. Twenty-three isolates without gyrA mutations were also found to exhibit reduced susceptibility to ofloxacin (MIC for 90% of strains ‫؍‬ 4 mg/liter), but largely remained susceptible to other drugs (MIC for 90% of strains < 1 mg/liter). Another 83 isolates without mutations were fully susceptible to all six fluoroquinolones (ofloxacin MIC for 90% of strains ‫؍‬ 1 mg/liter). In conclusion, high-level phenotypic resistance to fluoroquinolones among M. tuberculosis clinical isolates, which appears to be predominantly due to gyrA mutations, may be readily detected by genotyping techniques such as multiplex PCR amplimer conformation.Fluoroquinolones have antimycobacterial activities that possibly contribute a pivotal role in the second-line drug regimens used in the treatment of multidrug-resistant tuberculosis (32). However, in some communities, the resistance rates of Mycobacterium tuberculosis to these agents are surging (6, 9). To date, the genetic basis of this phenomenon has been attributed predominantly to mutations in the quinolone resistance-determining region of the gyrA gene (1,10,28,31).It would be of great relevance not only to study the genetic mutations responsible for fluoroquinolone resistance, but also to establish, as far as possible, their correlation with the resistance phenotypes of clinical isolates of M. tuberculosis. Such information facilitates rapid determination of the fluoroquinolone susceptibility status of the isolates and helps improve the clinical utility of these drugs in the settings of multidrug-resistant tuberculosis, as in cases involving streptomycin-and rifampin-resistant strains of M. tuberculosis (19,21). Hence, the major objective of this study was to identify the gyrA mutation(s), if any, in 138 clinical isolate...

Role of fluoroquinolones in the treatment of tuberculosis

“…Some studies have reported that M. tuberculosis strains containing D94G mutation in the gyrA gene exhibit high-level resistance to FQs while those with D94A mutation have lower MIC for LFX [93,96,97]. On the contrary, other reports have not found an association of different mutation patterns in gyrA or gyrB genes with the level of FQ resistance in M. tuberculosis isolates [89,94]. Several genotypic tests have recently been developed (Table III) for rapid (within 1-2 days) detection of vast majority of FQ-resistant clinical M. tuberculosis isolates [95].…”

“…The presence of missense mutations in the so-called quinolone resistance-determining region (QRDR) of gyrA or gyrB genes conferring resistance to FQs was subsequently confirmed by using clinical M. tuberculosis isolates in multiple studies [83][84][85][87][88][89][90][91][92][93][94][95][96][97]. The presence of S95 or T95 at gyrA codon 95 is a naturally occurring polymorphism that is not associated with resistance of M. tuberculosis isolates to FQs.…”

“…However, this polymorphism together with katG codon 463 polymorphism (R463 or L463), is used to define the three principal genetic groups of worldwide collection of M. tuberculosis strains [98][99][100]. The FQ-resistant clinical M. tuberculosis isolates more frequently contain mutations in gyrA gene, particularly at codon 90 (A90) or codon 94 (D94) but also occasionally at codon 74 (A74), codon 88 (G88) and codon 91 (S91) while mutations in few codons of the gyrB gene occur in a small minority of clinical strains [83,[89][90][91]. Surprisingly, some M. tuberculosis isolates have also been described that are hypersusceptible to FQs due to mutations at gyrA codon 80 (T80), particularly in association with other FQ resistance-associated mutations [83].…”

Schneller Nachweis von Mutationen im gyrA-Gen von Mycobacterium tuberculosis mit Relevanz für die Entstehung von Medikamentenresistenzen gegen Chinolone mittels der Real-Time PCR auf dem LightCycler® System

“…In (Jacobs 1999;Piddock 1999;Bryskier and Lowther 2002;Victor, van Helden et al 2002;Jacobs 2004) (Takiff, Salazar et al 1994;Cambau, Sougakoff et al 1994 (2); Alangaden, Manavathu et al 1995;Sullivan, Kreiswirth et al 1995;Kocagoz, Hackbarth et al 1996;Takiff, Cimino et al 1996;Williams, Chan et al 1996;Xu, Kreiswirth et al 1996;Perlman, El Sadr et al 1997;Sougakoff, Lemaitre et al 1997;Zhou, Dong et al 2000;Lee, Tang et al 2002;Siddiqi, Shamim et al 2002;Yew, Chan et al 2002;Johansen, Larsen et al 2003;Cheng, Yew et al 2004;Post, Willcox et al 2004;Giannoni, Iona et al 2005) (Ginsburg, Grosset et al 2003;Wade and Zhang 2004) …”

Clinical Laboratory Diagnostics for Mycobacterium tuberculosis

“…Other molecular assays that have been developed for the detection of resistance markers have included locked nucleic acid probes (van Doorn et al, 2008) and multiplex PCR amplimer conformation analysis (Cheng et al, 2004), pyrosequencing (Marttila et al, 2009;Garza-Gonzalez et al, 2010;Halse et al, 2010), oligonucleotide microarray (Caoili et al, 2006), and mass spectrometry (Wang et al, 2011).…”