Al-Mutairi Noura M, Ahmad Suhail, Mokaddas Eiman
Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait.
BMC Res Notes. 2011 Apr 14;4:123. doi: 10.1186/1756-0500-4-123.
Nearly 5% of all Mycobacterium tuberculosis strains worldwide are resistant at least to rifampicin and isoniazid (multidrug-resistant tuberculosis, MDR-TB). Inclusion of a fluoroquinolone and an injectable agent (kanamycin, amikacin or capreomycin) in multidrug therapy is crucial for proper treatment of MDR-TB. The incidence of MDR-TB in Kuwait is ~1%. MDR-TB strains additionally resistant to fluoroquinolones and injectable agents are defined as extensively drug-resistant (XDR-TB) strains and have been detected in >55 countries. Infections with XDR-TB strains have very poor prognosis. This study detected the occurrence of gyrA mutations associated with fluoroquinolone resistance among MDR-TB strains in Kuwait.
Direct DNA sequencing of quinolone resistance-determining region of gyrA gene was performed to detect fluoroquinolone resistance-associated mutations in 85 MDR-TB strains isolated from 55 TB patients and 25 pansusceptible M. tuberculosis strains. For isolates exhibiting gyrA mutations, 3'-end of rrs (16S rRNA) was sequenced for the detection of XDR-TB. Fingerprinting of fluoroquinolone resistant MDR-TB strains was performed by detecting mutations in three (81 bp hot-spot, N-terminal and cluster II) regions of rpoB, katG codon 315 and inhA-regulatory region, polymorphisms at gyrA codon 95 and katG codon 463 by DNA sequencing and by double-repetitive-element PCR for determining strain relatedness. None of the pansusceptible but six of 85 MDR-TB strains contained gyrA mutations. Only gyrA codon 94 was mutated in all six (D94A in one and D94G in five) strains. Three of six mutant strains were recovered from the same patient while three other strains represented individual patient isolates. Fingerprinting studies identified all individual patient isolates as epidemiologically distinct strains. All six strains with a gyrA mutation contained wild-type rrs sequence.
Although fluoroquinolones are generally not used for chemotherapy of TB and drug susceptibility testing for second-line drugs is not carried out in Kuwait, four of 55 (7%) individual patient MDR-TB strains contained mutations in gyrA gene. The data advocate routine drug susceptibility testing for this important second-line drug for proper management of MDR-TB in Kuwait. Lack of mutations in 3'-end of rrs gene that confer resistance to injectable agents reduce the likelihood of occurrence of XDR-TB, at present, in Kuwait.
全球近5%的结核分枝杆菌菌株至少对利福平及异烟肼耐药（耐多药结核病，MDR-TB）。在耐多药结核病的恰当治疗中，在多药治疗方案中加入氟喹诺酮类药物及一种注射用药物（卡那霉素、阿米卡星或卷曲霉素）至关重要。科威特耐多药结核病的发病率约为1%。对氟喹诺酮类药物及注射用药物额外耐药的耐多药结核菌株被定义为广泛耐药（XDR-TB）菌株，已在超过55个国家被检测到。感染广泛耐药结核菌株的预后非常差。本研究检测了科威特耐多药结核菌株中与氟喹诺酮耐药相关的gyrA基因突变的发生情况。
对从55例结核病患者分离出的85株耐多药结核菌株及25株全敏感结核分枝杆菌菌株进行gyrA基因喹诺酮耐药决定区的直接DNA测序，以检测与氟喹诺酮耐药相关的突变。对于表现出gyrA基因突变的分离株，对rrs（16S rRNA）的3′端进行测序以检测广泛耐药结核病。通过检测rpoB的三个区域（81 bp热点、N端和簇II）、katG密码子315和inhA调控区的突变、通过DNA测序检测gyrA密码子95和katG密码子463处的多态性以及通过双重复元件PCR来确定菌株相关性，对耐氟喹诺酮耐多药结核菌株进行指纹分析。所有全敏感菌株均未检测到gyrA基因突变，但85株耐多药结核菌株中有6株含有该突变。所有6株突变菌株中仅gyrA密码子94发生突变（1株为D94A，5株为D94G）。6株突变菌株中有3株来自同一患者，另外3株代表单个患者的分离株。指纹分析研究确定所有单个患者的分离株在流行病学上均为不同菌株。所有6株发生gyrA基因突变的菌株均含有野生型rrs序列。
尽管科威特一般不将氟喹诺酮类药物用于结核病化疗且未开展二线药物的药敏试验，但55例单个患者的耐多药结核菌株中有4株（7%）gyrA基因发生突变。这些数据支持对这种重要的二线药物进行常规药敏试验，以便在科威特对耐多药结核病进行恰当管理。目前在科威特，rrs基因3′端未发生赋予对注射用药物耐药性的突变，这降低了广泛耐药结核病发生的可能性。
