Mutations in gyrA and gyrB among Fluoroquinolone- and Multidrug-Resistant Mycobacterium tuberculosis Isolates

In order to correlate the mutations inside the entire gyrA and gyrB genes with the level of resistance to ofloxacin (OFX) and moxifloxacin (MFX) in isolates of multidrug-resistant Mycobacterium tuberculosis (MDR-TB), a total of 111 isolates were categorized into OFX-susceptible (MIC, ≤2 μg/ml) and low-level (MIC, 4 to 8 μg/ml) and high-level (MIC, ≥16 μg/ml) OFX-resistant isolates and MFX-susceptible (MIC, ≤0.5 μg/ml) and low-level (MIC, 1 to 2 μg/ml) and high-level (MIC, ≥4 μg/ml) MFX-resistant isolates. Resistance-associated mutations inside the gyrA gene were found in 30.2% of OFX-susceptible and 72.5% and 72.2% of low-level and high-level OFX-resistant isolates and in 28.6% of MFX-susceptible and 58.1% and 83.9% of low-level and high-level MFX-resistant isolates. Compared with OFX-susceptible isolates, low-level and high-level OFX-resistant isolates had a significantly higher prevalence of mutations at gyrA codons 88 to 94 (17.0%, 65.0%, and 72.2%, respectively; P < 0.001) and a higher prevalence of the gyrB G512R mutation (0.0%, 2.5%, and 16.7%, respectively; P = 0.006). Similarly, compared with MFX-susceptible isolates, low-level and high-level MFX-resistant isolates had a significantly higher prevalence of mutations at gyrA codons 88 to 94 (14.3%, 51.6%, and 80.6%, respectively; P < 0.001) as well as a higher prevalence of the gyrB G512R mutation (0.0%, 0.0%, and 12.9%, respectively; P = 0.011). D94G and D94N mutations in gyrA and the G512R mutation in gyrB were correlated with high-level MFX resistance, while the D94A mutation was associated with low-level MFX resistance. The prevalence of mutations at gyrA codons 88 to 94 and the gyrB G512R mutation were higher among fluoroquinolone (FQ)-susceptible East Asian (Beijing) and Indo-Oceanic strains than they were among Euro-American strains, implying that molecular techniques to detect FQ resistance may be less specific in areas with a high prevalence of East Asian (Beijing) and Indo-Oceanic strains.     

Prevalence and Molecular Characterization of Fluoroquinolone-Resistant Mycobacterium tuberculosis Isolates in China

China is one of the countries with the highest burdens of multidrug-resistant (MDR) and fluoroquinolone (FQ)-resistant tuberculosis (TB) globally. Nevertheless, knowledge about the prevalence and molecular characterization of FQ-resistant Mycobacterium tuberculosis isolates from this region remains scant. In this study, 138 M. tuberculosis isolates determined by the agar proportion susceptibility method to be resistant to ofloxacin (OFX) were enrolled from a national drug resistance survey of China. All these strains were tested for susceptibility to ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, and sparfloxacin using liquid Middlebrook 7H9 medium. The entire gyrA and gyrB genes conferring FQ resistance were sequenced, and spoligotyping was performed to distinguish different genotypes. Overall, the prevalence of resistance in China was highest for ofloxacin (3.76%), intermediate for levofloxacin (3.18%) and moxifloxacin (3.12%), and lowest for sparfloxacin (1.91%) and gatifloxacin (1.33%). Mutations in the gyrA gene were observed in 89 (64.5%) out of the 138 OFX-resistant M. tuberculosis strains. Positions 94 and 90 were the most frequent sites of mutation conferring FQ resistance on these strains, accounting for high-level FQ resistance. Furthermore, the Beijing genotype showed no association with high-level FQ resistance or distribution in hot spots in the quinolone resistance-determining region (QRDR) of gyrA. Our findings provide essential implications for the feasibility of genotypic tests relying on detection of mutations in the QRDR of gyrA and the shorter first-line treatment regimens based on FQs in China.     

Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in increased interest in the fluoroquinolones (FQs) as antituberculosis agents. To investigate the frequency and mechanisms of FQ resistance in M. tuberculosis, we cloned and sequenced the wild-type gyrA and gyrB genes, which encode the A and B subunits of the DNA gyrase, respectively; DNA gyrase is the main target of the FQs. On the basis of the sequence information, we performed DNA amplification for sequencing and single-strand conformation polymorphism analysis to examine the presumed quinolone resistance regions of gyrA and gyrB from reference strains (n = 4) and clinical isolates (n = 55). Mutations in codons of gyrA analogous to those described in other FQ-resistant bacteria were identified in all isolates (n = 14) for which the ciprofloxacin MIC was > 2 micrograms/ml. In addition, we selected ciprofloxacin-resistant mutants of Mycobacterium bovis BCG and M. tuberculosis Erdman and H37ra. Spontaneously resistant mutants developed at a frequency of 1 in 10(7) to 10(8) at ciprofloxacin concentrations of 2 micrograms/ml, but no primary resistant colonies were selected at higher ciprofloxacin concentrations. Replating of those first-step mutants selected for mutants with high levels of resistance which harbored gyrA mutations similar to those found among clinical FQ-resistant isolates. The gyrA and gyrB sequence information will facilitate analysis of the mechanisms of resistance to drugs which target the gyrase and the implementation of rapid strategies for the estimation of FQ susceptibility in clinical M. tuberculosis isolates.     

Molecular Analysis of the gyrA and gyrB Quinolone Resistance-Determining Regions of Fluoroquinolone-Resistant Clostridium difficile Mutants Selected In Vitro

Recent studies have suggested that exposure to fluoroquinolones represents a risk factor for the development of Clostridium difficile infections and that the acquisition of resistance to the newer fluoroquinolones is the major reason facilitating wide dissemination. In particular, moxifloxacin (MX) and levofloxacin (LE) have been recently associated with outbreaks caused by the C. difficile toxinotype III/PCR ribotype 027/pulsed-field gel electrophoresis type NAP1 strain. In this study, we evaluated the potential of MX and LE in the in vitro development of fluoroquinolone resistance mediated by GyrA and GyrB alterations. Resistant mutants were obtained from five C. difficile parent strains, susceptible to MX, LE, and gatifloxacin (GA) and belonging to different toxinotypes, by selection in the presence of increasing concentrations of MX and LE. Stable mutants showing substitutions in GyrA and/or GyrB were obtained from the parent strains after selection by both antibiotics. Mutants had MICs ranging from 8 to 128 μg/ml for MX, from 8 to 256 μg/ml for LE, and from 1.5 to ≥32 μg/ml for GA. The frequency of mutation ranged from 3.8 × 10⁻⁶ to 6.6 × 10⁻⁵ for MX and from 1.0 × 10⁻⁶ to 2.4 × 10⁻⁵ for LE. In total, six different substitutions in GyrA and five in GyrB were observed in this study. The majority of these substitutions has already been described for clinical isolates or has occurred at positions known to be involved in fluoroquinolone resistance. In particular, the substitution Thr82 to Ile in GyrA, the most common found in resistant C. difficile clinical isolates, was observed after selection with LE, whereas the substitution Asp426 to Val in GyrB, recently described in toxin A-negative/toxin B-positive epidemic strains, was observed after selection with MX. Interestingly, a reduced susceptibility to fluoroquinolones was observed in colonies isolated after the first and second steps of selection by both MX and LE, with no substitution in GyrA or GyrB. The results suggest a relevant role of fluoroquinolones in the emergence and selection of fluoroquinolone-resistant C. difficile strains also in vivo.Recent outbreaks of Clostridium difficile infections (CDI), with increased severity, high relapse rates, and significant mortality, have been related to the emergence of the hypervirulent C. difficile clone toxinotype III/PCR ribotype 027/pulsed-field gel electrophoresis type NAP1 (5, 23, 25-29, 31). Several studies have suggested that exposure to fluoroquinolones represents a risk factor for the development of CDI caused by C. difficile III/027/NAP1 and that the acquisition of resistance to the newer fluoroquinolones could have promoted its wide dissemination (6, 17, 30, 32-34).Fluoroquinolones are a family of broad-spectrum antibiotics extensively used in the treatment of a great variety of human infections. The in vitro activity of the older fluoroquinolones, such as ciprofloxacin, has been reported to be moderate or poor against anaerobes, including C. difficile (3, 8), whereas the third and the fourth generations of fluoroquinolones are characterized by improved activity against gram-positive cocci and anaerobic bacteria (19, 36). Fluoroquinolones act by inhibiting the action of DNA gyrase and topoisomerase IV, which are related but distinct enzymes involved in DNA synthesis (18). The mechanisms of resistance to fluoroquinolones in bacteria are basically two: (i) alterations in the targets of fluoroquinolones and (ii) decreased accumulation inside the bacteria due to impermeability of the membrane and/or an overexpression of efflux pump systems (19, 20, 36). The first mechanism of resistance is widespread in many bacteria, and it is due to amino acid substitutions in the quinolone-resistance determining region (QRDR) of the target enzymes (35). This is the principal mechanism of resistance also in C. difficile, and since, as already observed in other species, this bacterium does not have genes for topoisomerase IV, resistance is determined by alterations in the QRDR of either DNA gyrase subunit GyrA or GyrB (10, 38).Different amino acid substitutions have been identified in GyrA and GyrB in fluoroquinolone-resistant C. difficile strains. The most frequent is the amino acid change Thr82 to Ile in GyrA, which also characterizes the epidemic clone III/027/NAP1 (11, 38). Two other GyrA substitutions, Asp71 to Val and Ala118 to Thr, have been more rarely observed (1, 2, 10, 12, 38). Four different amino acid substitutions have been identified in GyrB: Arg447 to Lys, Arg447 to Leu, Asp426 to Asn, and Asp426 to Val (10, 11, 38). In particular, Asp426 to Val has been described in toxin A-negative/toxin B-positive C. difficile epidemic strains of recent isolation (11).In this study, we evaluated the potential of moxifloxacin (MX) and levofloxacin (LE), recently associated with outbreaks caused by C. difficile III/027/NAP1 (25, 31, 33), for the in vitro development of fluoroquinolone resistance mediated by GyrA and GyrB alterations in five different susceptible C. difficile strains. The sequence changes occurring in the QRDR of the derived fluoroquinolone-resistant mutants were analyzed and correlated with the in vitro resistance to MX, LE, and gatifloxacin (GA), another fluoroquinolone recently involved in C. difficile outbreaks (17, 33).     

Efficacy of Nitazoxanide against Clinical Isolates of Mycobacterium tuberculosis

Nitazoxanide (NTZ) has bactericidal activity against the H37Rv laboratory strain of Mycobacterium tuberculosis with a MIC of 16 μg/ml. However, its efficacy against clinical isolates of M. tuberculosis has not been determined. We found that NTZ''s MIC against 50 clinical isolates ranged from 12 to 28 μg/ml with a median of 16 μg/ml and was unaffected by resistance to first- or second-line antituberculosis drugs or a diversity of spoligotypes.     

结核分枝杆菌氟喹诺酮耐药与gyrA基因突变的关系

目的:了解结核分枝杆菌临床分离株氟喹诺酮类药物耐药与gyrA基因突变的关系.方法:选取经过全自动快速分枝杆菌培养鉴定药敏系统(BACTEC-MGIT960)检测的氟喹诺酮类药物敏感株30株,耐药株30株,针对其gyrA基因的氟喹诺酮类药物耐药决定区(QRDR)进行扩增,并将扩增产物进行T-A克隆后进行测序.结果:在这60株结核分枝杆菌临床分离株中,30株敏感株均未在QRDR检出有义突变,30株耐药株中有19株QRDR存在有义突变,主要突变位点为90、91和94.gyrA基因突变占结核分枝杆菌氟喹诺酮类药物耐药菌株的63.3％(19/30).结论:gyrA基因突变与结核分枝杆菌氟喹诺酮类药物耐药密切相关.     

耐氟喹诺酮类鲍曼不动杆菌Parc的变异研究

目的研究鲍曼不动杆菌拓扑异构酶ⅣC亚基(ParC)的变异与其耐氟喹诺酮类(FQNL)的关系。方法收集临床分离耐喹诺酮鲍曼不动杆菌30株及敏感株10株,测定其对萘啶酸、环丙沙星、左旋氧氟沙星的最低抑菌浓度(MIC),并对此30株菌ParC的基因(parC)进行PCR扩增和DNA序列的分析比较。结果在4株高度耐FQNL(MIC≥128 mg/L)和1株低度耐FQNL(MIC=64 mg/L)菌中存在ParC的变异,同FQNL耐药性相关的变异有丝氨酸Ser87(TCG)→Leu(TTG)。结论临床分离的鲍曼不动杆菌对喹诺酮类药物耐药的分子机制可表现为ParC基因87位氨基酸密码子突变,当鲍曼不动杆菌合并有ParC的变异时,其耐药性往往会增强。     

Systematic Analysis of Pyrazinamide-Resistant Spontaneous Mutants and Clinical Isolates of Mycobacterium tuberculosis

Pyrazinamide (PZA) is a first-line antitubercular drug known for its activity against persistent Mycobacterium tuberculosis bacilli. We set out to systematically determine the PZA susceptibility profiles and mutations in the pyrazinamidase (pncA) gene of a collection of multidrug-resistant tuberculosis (MDR-TB) clinical isolates and PZA-resistant (PZA(r)) spontaneous mutants. The frequency of acquired resistance to PZA was determined to be 10(-5) bacilli in vitro. Selection at a lower concentration of PZA yielded a significantly larger number of spontaneous mutants. The methodical approach employed allowed for determination of the frequency of the PZA(r) phenotype correlated with mutations in the pncA gene, which was 87.5% for the laboratory-selected spontaneous mutants examined in this study. As elucidated by structural analysis, most of the identified mutations were foreseen to affect protein activity through either alteration of an active site residue or destabilization of protein structure, indicating some preferential mutation site rather than random scattering. Twelve percent of the PZA(r) mutants did not have a pncA mutation, strongly indicating the presence of at least one other mechanism(s) of PZA(r).     

采用gyrB基因扩增快速鉴定结核分枝杆菌复合群的初步研究

目的:初步分析gyrB基因扩增用于结核分枝杆菌复合群(MTC)快速鉴定的可行性.方法:采用特异性引物进行分枝杆菌标准株和临床株的gyrB基因扩增,再通过琼脂糖凝胶电泳观察聚合酶链反应(PCR)结果.结果:15株各种分枝杆菌标准株中,结核分枝杆菌(MTB)、牛分枝杆菌和非洲分枝杆菌均扩增出1 184bp目的片段,而其余分枝杆菌PCR结果为阴性;94株分枝杆菌临床株中,60株MTB和10株牛分枝杆菌扩增阳性.而其他分枝杆菌(包括6株脓肿分枝杆菌、5株偶发分枝杆菌、4株龟分枝杆菌、3株鸟分枝杆菌、3株胞内分枝杆菌、2株耻垢分枝杆菌、1株堪萨斯分枝杆菌)均扩增阴性.结论:采用针对MTC的特异性引物进行gyrB基因扩增能快速、准确地鉴别MTC和非结核分枝杆菌.     

Topoisomerase Mutations in Fluoroquinolone-Resistant and Methicillin-Susceptible and -Resistant Clinical Isolates of Staphylococcus aureus

The incidence of the various mutations in the genes encoding topoisomerase IV and DNA gyrase in fluoroquinolone-resistant clinical isolates of Staphylococcus aureus is not known. Using restriction fragment length polymorphism analysis and DNA sequencing, we found that in fluoroquinolone- and methicillin-resistant strains, mutations in grlA and gyrA are quite likely to be present together. For fluoroquinolone-resistant but methicillin-susceptible strains, mutations in grlA alone are more common.     

Characterization of Recombinant Fluoroquinolone-Resistant Pneumococcus-Like Isolates

Fourteen fluoroquinolone-resistant streptococcal isolates with recombinant DNA topoisomerase genes, preliminarily identified as pneumococci, were further characterized using phenotypic and genotypic approaches. Phenotypic tests classified them as atypical pneumococci. Phylogenetic relationships were analyzed by using the sequences of seven housekeeping alleles from these isolates and from isolates of Streptococcus pneumoniae, Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae. Four isolates grouped with S. pneumoniae, seven grouped with S. pseudopneumoniae, and three grouped with S. mitis. These results generally agreed with those obtained with an optochin susceptibility test and with the organization of the atp operon chromosomal region, encoding the F_oF₁ H⁺-ATPase (the target of optochin). All seven isolates grouping with S. pseudopneumoniae share the same spr1368-atpC-atpA gene order; all four grouping with S. pneumoniae share the spr1368-IS1239-atpC-atpA order, and two out of the three grouping with S. mitis share the spr1284-atpC-atpA order. In addition, evidence for recombination within the seven housekeeping alleles of the S. pseudopneumoniae population was provided by several methods: the index of association (0.4598, P < 0.001), the pairwise homoplasy index, and the split-decomposition method. This study confirms the existence of pneumococci among the alpha-hemolytic streptococci with DNA topoisomerase genes showing a mosaic structure and reveals a close relationship between atypical pneumococci and S. pseudopneumoniae.     

Plasmid-mediated complementation of gyrA and gyrB in fluoroquinolone-resistant Bacteroides fragilis

OBJECTIVES: To identify whether mutations in gyrA and gyrB confer fluoroquinolone resistance in Bacteroides fragilis. METHODS: Eight fluoroquinolone-resistant (FQR) strains were complemented with plasmid-mediated B. fragilis wild-type gyrA (pMP1) and gyrB (pMP2), and MICs determined. Sequence analysis of the gyrA and gyrB quinolone resistance determining region (QRDR) was performed for all strains. RESULTS: MICs of fluoroquinolones were two- to 32-fold higher than wild-type for all mutants. Five mutants had a substitution in GyrA (Ser-82-->Phe), one mutant had a substitution in GyrA (Asp-81-->Gly), one mutant had a substitution in GyrB (Glu-478-->Lys), and one resistant strain did not contain mutations in the QRDR of gyrA or gyrB. Following complementation with pMP1 or pMP2, the MICs of fluoroquinolones were reduced two- to 32-fold for the mutants. CONCLUSION: These studies verify that substitutions in GyrA and GyrB confer resistance in B. fragilis. Other mechanisms are also responsible for resistance since not all resistant strains fully complemented to the wild-type phenotype.     

耐左氧氟沙星结核分枝杆菌gyrA基因上新的突变位点

目的:分析耐左氧氟沙星结核分枝杆菌临床菌株gyrA基因的突变情况及其耐药机制.方法:用聚合酶链反应(PER)和DNA直接测序技术(DS)测定64株耐左氧氟沙星结核分枝杆菌gyrA基因的QRDR(quinolone resistance-determining regions)序列.结果:64株耐药菌株有47株QRDR序列发生突变,其中45株为单位点突变,另2株为双位点突变;突变分布为70位突变2株、89位1株、90位12株、91位4株、94位30株,其中70位和89位为新发现的突变位点.结论:结核分枝杆菌喹诺酮类药物耐受现象与gyrA基因QRDR的突变有关,包括新发现的70位和89位突变.     

结核分枝杆菌耐氧氟沙星临床分离株gyrA基因突变的研究

目的了解结核分枝杆菌(MTB)临床分离株对氧氟沙星的耐药性与gyrA基因突变的关系。方法对20株随机筛选的耐氧氟沙星MTB临床分离株行gyrA基因喹诺酮类药物耐药决定区(QRDR)序列测定。结果18株发现了有义突变,其中2株第91位密码子由TCG(丝氨酸)→CCG(脯氨酸),3株第90位密码子由CCG(甘氨酸)→GTG(缬氨酸),10株第94位密码子由GAC(天冬氨酸)→GGC(赖氨酸),2株第94位密码子由GAC(天冬氨酸)→GCC(甘氨酸),1株第94位密码子由GAC(天冬氨酸)→GTC(缬氨酸)。6例既往未应用过氟喹诺酮类药物抗结核治疗,2例曾在结核诊断前应用氟喹诺酮类药物经验性抗感染治疗1周,12例曾应用氟喹诺酮类药物抗结核治疗。结论gyrA基因突变是MTB对氟喹诺酮类药物产生耐药的机制之一;gyrA基因的有义突变主要发生在第90位、91位、94位密码子上。     

gyrA and gyrB mutations in quinolone-resistant strains of Escherichia coli.

The proportion of gyrA and gyrB mutations in quinolone-resistant Escherichia coli strains was examined by introducing cloned wild-type gyrA and gyrB genes. In 25 spontaneous mutants of strain KL16, 13 had gyrA and 12 had gyrB mutations. In eight clinical isolates, five had gyrA mutations and one had a gyrB mutation; mutations in two isolates remained unidentified.     

Comparison of Different Drug Susceptibility Test Methods To Detect Rifampin Heteroresistance in Mycobacterium tuberculosis

We compared the efficiencies of different drug susceptibility testing methods in detecting rifampin (RIF) heteroresistance in Mycobacterium tuberculosis. Our data revealed that the broth dilution method found more resistance than MGIT did (P = 0.046) for the low-resistance group. Similarly, the broth dilution method was more sensitive in detecting RIF heteroresistance in subpopulations with low growth rates than was MGIT (P = 0.033). In conclusion, our data demonstrated that the broth dilution method was more sensitive than MGIT in detecting RIF heteroresistance.