Mutations In gyrA Genes Research Articles

Molecular analysis of low-level fluoroquinolone resistance in clinical isolates of Moraxella catarrhalis.

We investigated antimicrobial susceptibility and the molecular mechanism underlying low-level resistance to fluoroquinolones in 70 non-duplicate clinical isolates of Moraxella catarrhalis. The isolates were collected in a general hospital in Tokyo, Japan, between January and October 2013 from 38 men and 32 women; most of the isolates (48 out of 70, 68.5%) were obtained from post-nasal drips of children. The antimicrobial susceptibility of M. catarrhalis isolates was determined with an Etest, and low-level fluoroquinolone-resistant isolates were subtyped by PFGE. Mutations in the gyrA and parC genes were determined by PCR and sequencing. PCR products of the gyrA and parC genes from the low-level fluoroquinolone-resistant isolates were transformed into a fluoroquinolone-susceptible strain. Among the 70 isolates, five (7.1%) exhibited elevated fluoroquinolone MICs (levofloxacin, 1.0 mg l(-1); ciprofloxacin, 0.5 mg l(-1)) and different PFGE patterns. The patients from whom these five isolates were isolated had not undergone treatment with fluoroquinolones for the past 6 months. Each of the five low-level fluoroquinolone-resistant isolates had a gyrA gene mutation resulting in a Thr-to-Ile substitution at aa 80 (T80I) in the GyrA protein, while no changes were detected in the parC gene. A transformant carrying the gyrA gene containing the T80I substitution, which corresponded to Ser83 in Escherichia coli, displayed an elevated fluoroquinolone MIC and contained the T80I alteration in GyrA. Thus, our findings reveal that the low-level resistance to fluoroquinolones in M. catarrhalis is due to an amino acid substitution of Thr80 to Ile in GyrA. This is the first evidence of low-level fluoroquinolone resistance in M. catarrhalis.

A novel gyrB gene mutation in fluoroquinolone resistant clinical isolates of Mycobacterium tuberculosis

Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis can be conferred by mutations in gyrA or gyrB gene. Recent studies suggest that amino acid substitutions in gyrB gene may also play a crucial role in resistance, but genetic studies of these mutations in M. tuberculosis are lacking.

Pyrosequencing for rapid detection of tuberculosis resistance in clinical isolates and sputum samples from re-treatment pulmonary tuberculosis patients.

BackgroundMultidrug-resistant tuberculosis (MDR-TB) is a major public health problem. Early diagnosis of MDR-TB patients is essential for minimizing the risk of Mycobacterium tuberculosis (MTB) transmission. The conventional drug susceptibility testing (DST) methods for detection of drug-resistant M.tuberculosis are laborious and cannot provide the rapid detection for clinical practice.MethodsThe aim of this study was to develop a pyrosequencing approach for the simultaneous detection of resistance to rifampin (RIF), isoniazid (INH), ethambutol (EMB), streptomycin (SM), ofloxacin (OFL) and amikacin (AMK) in M. tuberculosis clinical isolates and sputum samples from re-treatment pulmonary tuberculosis (PTB) patients. We identified the optimum conditions for detection mutation of rpoB, katG, rpsl, embB, gyrA and rrs gene by pyrosequencing. Then this approach was applied to detect 205 clinical isolates and 24 sputum samples of M. tuberculosis from re-treatment PTB patients.ResultsThe mutations of rpoB and gyrA gene were detected by pyrosequencig with the SQA mode, and the mutations of katG, rpsl, embB, gyrA and rrs gene were detected by pyrosequencing with SNP mode. Compared with the Bactec MGIT 960 mycobacterial detection system, the accuracy of pyrosequencing for the detection of RIF, INH, EMB, SM, AMK and OFL resistance in clinical isolates was 95.0%, 79.2%, 70.3%, 84.5%, 96.5% and 91.1%, respectively. In sputum samples the accuracy was 83.3%, 83.3%, 60.9%, 83.3%, 87.5% and 91.7%, respectively.ConclusionsThe newly established pyrosequencing assay is a rapid and high-throughput method for the detection of resistance to RIF, INH, SM, EMB, OFL and AMK in M.tuberculosis. Pyrosequencing can be used as a practical molecular diagnostic tool for screening and predicting the resistance of re-treatment pulmonary tuberculosis patients.

Triple Therapy with High‐Dose Proton‐Pump Inhibitor, Amoxicillin, and Doxycycline Is Useless for Helicobacter pylori Eradication: A Proof‐of‐Concept Study

Helicobacter pylori resistance to antibiotics is steadily increasing and multidrug-resistant strains are common and difficult to eliminate, mainly in countries where bismuth, tetracycline, furazolidone, and rifabutin are unavailable. To evaluate the efficacy and safety of a triple therapy with proton-pump inhibitor (PPI), amoxicillin, and doxycycline in patients with multidrug-resistant H. pylori. This prospective study involved 16 patients (13 females; mean age - 50 ± 11.3 years) infected by H. pylori with known resistance to clarithromycin, metronidazole, and levofloxacin, but susceptibility to amoxicillin and tetracycline. All patients were previously submitted to upper endoscopy with gastric biopsies for H. pylori culture and susceptibility testing by Etest. Mutations in 23S rRNA and gyrA genes were determined by real-time PCR. A 10-day eradication regimen with PPI (double-standard dose b.i.d.), amoxicillin (1000 mg b.i.d.), and doxycycline (100 mg b.i.d.) was prescribed after pretreatment with PPI during 3 days. Eradication success was assessed by (13) C-urea breath test 6-10 weeks after treatment. Compliance and adverse events were determined through phone contact immediately after treatment and specific written questionnaires. Only one patient did not complete treatment due to adverse events. Another four patients experienced mild side effects not affecting compliance. The control (13) C-urea breath test was positive in all patients. Per-protocol and intention-to-treat eradication rates were 0%. Although safe, a triple-therapy protocol with high-dose PPI, amoxicillin, and doxycycline is useless for multidrug-resistant H. pylori eradication.

Analysis of quinolone-resistance in commensal and diarrheagenic Escherichia coli isolates from infants in Lima, Peru

Antibiotic resistance is an increasing problem, particularly in countries where antibiotic use is frequently not controlled. The aim of this study was to analyse the prevalence of the molecular mechanisms of quinolone-resistance in E. coli isolated from faeces of healthy Peruvian children or those presenting diarrhoea. The presence of target mutations, transferable quinolone-resistance mechanisms and the role of Phe-Arg-β-Naphtylamyde inhibitible efflux pumps were studied in 96 Escherichia coli (46 diarrheogenic and 50 non-diarrheogenic) isolates exhibiting resistance or diminished susceptibility to quinolones. The most resistant phenotype, Nal(R) and Cip(R), was most frequently present in isolates of healthy children. The distribution of quinolone resistance mechanisms between diarrheogenic (DEC) and commensal (non DEC) isolates was equitable, although the aac(6')Ib-cr gene was mainly detected in DEC isolates: 17 (34%) vs non DEC isolates nine (20%). QnrB was present in five (10%) DEC vs three (6%) non DEC isolates. Point mutations in gyrA and parC genes play a relevant role in quinolone resistance acquisition and highlight the role of efflux pumps also. This study provides knowledge about the molecular mechanisms involved in quinolone resistance in isolates in a non exposed population under high community antibiotic pressure.

Characterization of multiple and extensively drug resistant Mycobacterium tuberculosis isolates with different ofloxacin-resistance levels

The goal of the study was to determine different mutation types in gyrA and gyrB genes in Mycobacterium tuberculosis strains with low-level (2 μg/ml) and high-level (10 μg/ml) ofloxacin (OFL) resistance and to compare genetic diversity of ofloxacin-resistant and susceptible M. tuberculosis isolates. M. tuberculosis isolates were collected in Leningrad Region in 2011. DNA sequencing showed that 54.3% of isolates with low-level and 76.9% of isolates with high-level OFL-resistance had mutations in gyrA gene. Few isolates carried mutations in gyrB gene - five among isolates with low-level resistance and two among high-level resistant isolates. Altogether, detection of point mutations in both DNA gyrase genes allows to identify 66.9% of mycobacterial isolates with low-level and 84.5% of isolates with high-level of OFL-resistance. Novel mutations S91L in gyrA gene and S512L in gyrB gene described in this study were detected in OFL-resistant isolates and may play role in M. tuberculosis fluoroquinolone resistance. M. tuberculosis Beijing family spoligotypes were identified among 70.8% of isolates with low-level resistance, 84.6% of isolates with high-level resistance and 50% of strains susceptible to all tuberculosis drugs. Fisher's exact test revealed significant difference between Beijing prevalence in groups of drug-susceptible or high-level OFL-resistant M. tuberculosis strains (p-value = 0.032).

Expression of the marA, soxS, acrB and ramA genes related to the AcrAB/TolC efflux pump in Salmonella enterica strains with and without quinolone resistance-determining regions gyrA gene mutations

Several studies have been conducted in recent years to elucidate the structure, function and significance of AcrB, MarA, SoxS and RamA in Salmonella enterica. In this study, the relative quantification of acrB, soxS, marA and ramA genes expression was evaluated in 14 strains of S. enterica, with or without accompanying mutations in the quinolone resistance-determining regions of the gyrA gene, that were exposed to ciprofloxacin during the exponential growth phase. The presence of ciprofloxacin during the log phase of bacterial growth activated the genes marA, soxS, ramA and acrB in all S. enterica strains analyzed in this study. The highest expression levels for acrB were observed in strains with gyrA mutation, and marA showed the highest expression in the strains without mutation. Considering only the strains with ciprofloxacin minimum inhibitory concentration values<0.125μg/mL (sensitive to ciprofloxacin), the most expressed gene in the strains both with and without mutations was acrB. In the strains with ciprofloxacin minimum inhibitory concentration values≥0.125μg/mL (low susceptibility), with and without mutations in gyrA, the most expressed gene was marA. In this study, we observed that strains resistant to nalidixic acid may express genes associated with the efflux pump and the expression of the AcrAB-TolC pump genes seems to occur independently of mutations in gyrA.

Molecular basis of high-level ciprofloxacin resistance in Neisseria gonorrhoeae strains from Shandong Province, China

In the study, the ciprofloxacin resistance rate was 100%. High-level ciprofloxacin resistance rate was 63.55%. Sixteen different mutation patterns involved in the formation of ciprofloxacin resistance were identified. The most prevalent were patterns P7 (25.2%), P8 (15.0%), P9 (11.2%), P1 (10.3%), and P5 (10.3%). All of the 107 NG isolates analyzed for mutations in the study have demonstrated a change of Ser-91 → Phe in the gyrA gene, and all except one have demonstrated a change in position 95 of the amino acid sequence. All of the 68 high-level QRNG isolates had double mutations in gyrA gene combined with a single or two mutations in parC gene. It is most important that a new mutation site of Ile-97 → Met in gyrA and a new mutation of Leu-106 → Ile in parC were found in the study, both leading to high-level ciprofloxacin resistance (MIC values, 8 μg/mL, 32 μg/mL, respectively). Therefore, we confim that gyrA mutations are necessary for the fluoroquinolone resistance phenotype and parC mutations are correlated intimately with high-level fluoroquinolone resistance. In China fluoroquinolone resistance in Neisseria gonorrhoeae strains is very serious and the new mutation sites in the fluoroquinolone resistance-determining regions emerge more and more quickly. Hence, in China fluoroquinolones, which are used to treat gonorrhoea presently, should be substituted by a new antibiotics.

Multiple-locus variable number tandem repeat analysis of Neisseria gonorrhoeae isolates in Russia

In the present study, a multiple-locus variable number tandem repeat analysis (MLVA) was used to assess the molecular epidemiology of Neisseria gonorrhoeae clinical isolates originating from different regions of Russia. MLVA, based on seven loci, was performed on 218 isolates that were previously tested for susceptibility to penicillin, tetracycline and ciprofloxacin and for the presence of certain genetic determinants of drug resistance. In total, 83 MLVA types were identified, indicating that MLVA is a highly discriminatory technique with a Hunter–Gaston discriminatory index of 0.963 (95% CI, 0.950–0.977). MLVA type 16 was shown to be the most prevalent type and is undoubtedly associated with a multidrug resistant phenotype. The spread of MLVA type 16 from Moscow to Irkutsk suggests that this type has a highly successful transmission rate. Hierarchical cluster analysis of the MLVA profiles classified 208 isolates (95%) into six large groups (containing more than 10 isolates). Clusters differed in geographical characteristics and susceptibility profiles. MLVA cluster A comprised in total 34 isolates and was unambiguously associated with multidrug resistance. Most isolates in cluster A carried mutations in penA, ponA, rpsJ, mtrR, gyrA, and parC genes. MLVA cluster D was associated with resistance to penicillin and with mutations in ponA and rpsJ genes and the presence of plasmid-borne bla(TEM-1) gene. MLVA clusters B, C and E were associated with susceptibility to ciprofloxacin and had a lack of mutations in ponA, rpsJ, gyrA, and parC genes. We conclude that MLVA will be a useful tool for N. gonorrhoeae epidemiological studies.

Plasmid-mediated quinolone resistance mechanisms in ESBL positive Escherichia coli and Klebsiella pneumoniae strains at a Tertiary-Care Hospital in Turkey

Plasmid-mediated quinolone resistance mechanisms in extended spectrum beta-lactamase positive and quinolone resistant Escherichia coli and Klebsiella pneumoniae strains isolated from Ege University Hospital were investigated. The presence of qnrA, qnrB, qnrS, aac(6’)-Ib and qepA genes were detected by PCR and the products were sequenced. Clonal relationship of isolates was determined by REP-PCR and mutations in gyrA and parC genes were investigated in representative strains. aac(6’)-Ib-cr, qnrB and qnrS genes were detected in both E. coli and K. pneumoniae strains, but qnrA detected only in K. pneumoniae strains. qepA determinant is detected in an E. coli strain first time in Turkey. Mutations were observed in both gyrA and parC genes of all representative nalidixic acid and ciprofloxacin resistant E. coli isolates but no mutation was found in parC genes of E. coli and K. pneumoniae strains that were resistant to only nalidixic acid.

Comparison of the Virulence Potential of Acinetobacter Strains from Clinical and Environmental Sources

Several Acinetobacter strains have utility for biotechnology applications, yet some are opportunistic pathogens. We compared strains of seven Acinetobacter species (baumannii, Ab; calcoaceticus, Ac; guillouiae, Ag; haemolyticus, Ah; lwoffii, Al; junii, Aj; and venetianus, Av-RAG-1) for their potential virulence attributes, including proliferation in mammalian cell conditions, haemolytic/cytolytic activity, ability to elicit inflammatory signals, and antibiotic susceptibility. Only Ah grew at 102 and 104 bacteria/well in mammalian cell culture medium at 37°C. However, co-culture with colonic epithelial cells (HT29) improved growth of all bacterial strains, except Av-RAG-1. Cytotoxicity of Ab and Ah toward HT29 was at least double that of other test bacteria. These effects included bacterial adherence, loss of metabolism, substrate detachment, and cytolysis. Only Ab and Ah exhibited resistance to killing by macrophage-like J774A.1 cells. Haemolytic activity of Ah and Av-RAG-1 was strong, but undetectable for other strains. When killed with an antibiotic, Ab, Ah, Aj and Av-RAG-1 induced 3 to 9-fold elevated HT29 interleukin (IL)-8 levels. However, none of the strains altered levels of J774A.1 pro-inflammatory cytokines (IL-1β, IL-6 and tumor necrosis factor-α). Antibiotic susceptibility profiling showed that Ab, Ag and Aj were viable at low concentrations of some antibiotics. All strains were positive for virulence factor genes ompA and epsA, and negative for mutations in gyrA and parC genes that convey fluoroquinolone resistance. The data demonstrate that Av-RAG-1, Ag and Al lack some potentially harmful characteristics compared to other Acinetobacter strains tested, but the biotechnology candidate Av-RAG-1 should be scrutinized further prior to widespread use.

Cornmeal-induced resistance to ciprofloxacin and erythromycin in enterococci

ObjectiveTo establish a model ecosystem to study the impact of cornmeal on the appearance and persistence of the erythromycin (ERY)- and ciprofloxacin (CIP)-resistant phenotypes in waterborne enterococci. MethodsAfter the model ecosystem was established, the system was divided into six dose groups, with the addition of 8, 4, 1, 0.25, 0.05, and 0gL−1 sterilized cornmeal. System mud samples were collected at 0, 1, 3, 7, 14, 30, 40, 61, and 130d, and enterococci present in the mud samples were evaluated for their sensitivities to CIP and ERY. PCR was employed to detect genes such as gyrA and ermB. The gyrA gene was sequenced, and codons 83 and 87 were analyzed for mutations. Results(1) The addition of 0.05–8gL−1 cornmeal had an impact on CIP resistance. The higher the dose of cornmeal added, the larger the impact it generated. Furthermore, the earlier the emergence of CIP-resistant strains, the greater the incidence of drug resistance. The impact of cornmeal on resistance to ERY was less consistent, and the degree of the impact was not in proportion to the dose of cornmeal added. (2) There were no mutations at codons 83 and 87 in the gyrA genes from 102 strains isolated from the model ecosystem. The incidence of ermB-positive strains of ERY-resistant enterococci (28 strains) was 78.6%, and the incidence of ermB-positive strains of ERY-sensitive enterococci (16 strains) was 0%. Conclusions(1) Adding different doses of cornmeal can facilitate resistance to CIP and ERY in waterborne enterococci. In this study, the degree of resistance was related to the cornmeal dose. (2) In the model ecosystem, enterococcal CIP resistance was not caused by a gyrA gene mutation; however, in the vast majority of cases, resistance to ERY was related to the ermB resistance gene.

Gemifloxacin Can Partially Overcome Quinolone Resistance of H. pylori with gyrA Mutation in Taiwan

The levofloxacin resistance caused by gyrA gene mutation is rising rapidly to limit wide application for Helicobacter pylori eradication. We investigated whether gemifloxacin has a superior antimicrobial activity to levofloxacin against H. pylori. Forty-four consecutive clinical H. pylori isolates with levofloxacin resistance and 80 randomly selected levofloxacin-sensitive controls were tested for gemifloxacin sensitivity by E-test. The resistance to levofloxacin or gemifloxacin was defined as minimal inhibitory concentration (MIC) > 1 mg/L. The clinical features and GyrA mutation patterns checked by direct sequencing were also analyzed to assess its association with the H. pylori gemifloxacin resistance. All levofloxacin-sensitive H. pylori isolates were sensitive to gemifloxacin. Eight strains (18.2%) resistant to levofloxacin could be still sensitive to gemifloxacin. Gemifloxacin achieved a 5-time lower in MIC levels against levofloxacin-resistant isolates. Nearly all levofloxacin-resistant isolates (97.7%, 43/44) had GyrA mutation at amino acid position 87 or 91. Double mutation sites may play dual roles in quinolone resistance, as N87K plus H57Y or D91N plus V77A mutations showed high-level resistance to both quinolones; whereas D91Y plus A97V or D91N plus A97V mutations showed low level levofloxacin resistance to become sensitive to gemifloxacin. In H. pylori isolates with single N87K, D91Y or D91N mutation, near 20% was gemifloxacin-sensitive and levofloxacin-resistant. The gemifloxacin-resistant rate of H. pylori was higher in patients with gastric ulcer than in those without (p <.05). Gemifloxacin is superior to levofloxacin in antimicrobial activity against clinical H. pylori isolates, and even overcome some levofloxacin resistance.

Molecular epidemiology of fluoroquinolone resistance in invasive clinical isolates of Streptococcus pneumoniae in Seville

Due to the emergence of drug-resistant pneumococcal isolates, new fluoroquinolones have been recommended for the treatment of pneumococcal infections. The purpose of this study was to establish surveillance, and to conduct molecular characterization, of fluoroquinolone-resistant Streptococcus pneumoniae in Seville. Norfloxacin-resistant S. pneumoniae isolates were characterized by quinolone resistance-determining region (QRDR) substitutions, reserpine-sensitive efflux, serotype and by pulsed-field gel electrophoresis (PFGE) patterns. Fourteen isolates (5.1%) showed an MIC>16 μg/ml to norfloxacin. Eight of 10 adult isolates were susceptible to levofloxacin. The 4 infant isolates with norfloxacin MIC>16 μg/ml were susceptible to levofloxacin. Seven of these 12 low-level-resistant isolates had mutations in ParC, while mutations both in ParC and GyrA genes were only detected in one of the two high-level-resistant isolates. All the isolates without QRDR substitutions that remained norfloxacin-resistant were positive for reserpine-inhibited efflux. The serotyping and PFGE revealed significant heterogeneity. We obtained 9 different profiles, 3 of which had two isolates each. Two of the isolates with the same pulsotype were from the same patient. The first isolate showed a mutation in the QRDR of ParC, and the second one had an additional GyrA mutation. In our study a levofloxacin resistance rate of 0.7% was found among invasive isolates. Although resistance level is low, surveillance is necessary, especially to prevent cases of in vivo resistance development as reported.

English

Multidrug resistant strains of&nbsp;Staphylococcus aureus&nbsp;cause serious problems worldwide. We have characterized the quinolone resistant mechanisms in clinical isolates of&nbsp;S. aureus&nbsp;and tested their susceptibilities to other classes of antibiotics. Results of this study showed high level of resistance to quinolone in the selected isolates and suggested that resistance is due to mutations in&nbsp;gyrA&nbsp;and&nbsp;parC&nbsp;genes in addition to efflux mechanism. The selected strains were not only resistant to quinolones but also found resistant to a number of other antibiotics. This study also shed some light on the possibility of using Clove as an efflux pump inhibitor. The minimum inhibitory concentration of ciprofloxacin against multidrug resistant isolates was reduced from 256 to 64 &micro;g/ml when used in combination with Clove extract. This result indicated that Clove is a potential efflux pump inhibitor. In addition, the effect of different efflux pump inhibitors on the resistance of these isolates was determined. &nbsp; Key words:&nbsp;Staphylococcus aureus,&nbsp;fluoroquinolone antibiotics, ciprofloxacin, efflux pump inhibitor, clove. .

Detection of Mutation in gyrA and parC Gene on Resistant Salmonella enterica Serovars. Isolated from Two Hospitals in South East Nigeria

Detection of Mutation in gyrA and parC Gene on Resistant Salmonella enterica Serovars. Isolated from Two Hospitals in South East Nigeria

Mutations ingyrAandparCGenes and Plasmid-Mediated Quinolone Resistance in Non-typhoidSalmonellaIsolated from Pediatric Patients with Diarrhea in Seoul

A total of 91 non-typhoid Salmonella isolated from pediatric patients with diarrhea in Seoul from 2003 to 2009 was tested for antimicrobial susceptibility of nalidixic acid (NA). Forty strains of NA resistance or intermediate susceptible non-typhoid Salmonella were identified and their minimum inhibitory concentrations (MICs) of NA, ciprofloxacin (CIP), and norfloxacin (NOR) were determined. Of the 40 isolates, 26 were resistant to NA (MIC >256 μg/ml). Only one isolate was high-level resistant to CIP (12 μg/ml) and NOR (48 μg/ml). Mutations in gyrA and parC genes were studied by PCR and sequencing. All NA-resistant isolates carried point mutations in the gyrA quinolone resistance determining regions (QRDR) at codon 83 or 87 (MICs of NA, >256 μg/ml; MICs of CIP, 0.047~0.25 μg/ml; MICs of NOR, 0.38~1.5 μg/ml). A double change in GyrA was found in one Salmonella Enteritidis (MIC of CIP, 12 μg/ml; MIC of NOR, 48 μg/ml). In respect of the ParC protein, a single change at Thr57→Ser was found in 3 isolates (MICs of NA, >256 μg/ml; MICs of CIP, 0.19~0.25 μg/ml; MICs of NOR, 1 μg/ml). At the same time, these strains changed from Ser83 to Tyr in the gyrA. The result of the investigation for the prevalence of plasmid-mediated quinolone resistance (PMQR) genes, 14 isolates harbored qnr gene among 40 isolates. All of 14 isolates showed decreased susceptibility at NA (MICs 4~16 μg/ml) and except one strain, all of qnr genes were identified as qnrB. Mutations in the gyrA gene and production of PMQR determinants were critical for quinolone resistance and decreased susceptibility to fluoroquinolone in these isolates.

Genetic Classification and Antimicrobial Resistance ofUreaplasma urealyticumIsolated from Urine

Recently, polymerase chain reaction (PCR)-based methods have been used to reclassify Ureaplasma urealyticum into two independent species (spp.), designating U. parvum and U. urealyticum. In the current study, we aim to reclassify U. urealyticum and to analyze the correlation between the presence of a genetic marker and an antibiotic resistance of U. urealyticum. Susceptibility test against tetracycline, levofloxain or moxifloxacin was performed by broth microdilution method. The presence of tet(M) gene and the mutations of quinolone resistance-determining regions (QRDRs) were analyzed by PCR and sequencing. Among fourteen Ureaplasma isolates, three were identified as U. parvum and eleven were identified as U. urealyticum, and this is first report showing that two independent spp. of U. urealyticum isolated from Korean are present. The minimum inhibitory concentration (MIC) ranges for Ureaplasma isolates were as follows: tetracycline 0.25~128 μg/ml, levofloxacin 1~8 μg/ml, and moxifloxacin 0.5~4 μg/ml. The tet(M) determinant was found in 3 among 14 Ureaplasma isolates with tetracycline MIC of >16 μg/ml, suggesting that the presence of the tet(M) determinant is associated with tetracycline resistance. Mutations of gyrA, gyrB, parC, and parE genes in the QRDRs were found in 3 among 14 Ureaplasma isolates, exhibiting only parE gene mutation is associated with fluoroquinolone resistance.

Multiplex allele-specific PCR combined with PCR-RFLP analysis for rapid detection of gyrA gene fluoroquinolone resistance mutations in Mycobacterium tuberculosis

A combined use of MAS-PCR (multiplex allele-specific PCR) and PCR-RFLP (PCR restriction fragment length polymorphism), was established to detect mutations in codons 90, 91 and 94 of the gyrA gene in Mycobacterium tuberculosis ( M. tuberculosis). With conventional phenotypic drug susceptibility testing as a reference standard, the sensitivity, specificity and accuracy of the modified method for gyrA gene mutation detection were 70.8%, 100% and 84.8% respectively.

GyrAandgyrBGene Mutation in Ciprofloxacin-ResistantMycobacterium massilienseClinical Isolates from Southern Brazil

Fluoroquinolones (FQs) have been increasingly used for effective treatment of infections caused by rapidly growing mycobacteria, and resistance to this drug has been predominantly attributed to gyrA and gyrB mutations. Accordingly, this study investigated a total of 36 Mycobacterium massiliense clinical isolates for their susceptibility to ciprofloxacin and presence of gyrA and gyrB gene mutations. The minimal inhibitory concentration (MIC) values, determined by broth microdilution method, of 35 ciprofloxacin-resistant isolates ranged between 4 and 16 μg/mL and a single susceptible isolate was obtained. A total of 31 of 35 (88.5%) ciprofloxacin-resistant isolates presented an amino acid substitution at codon 90 (Ala-90→Val) and no isolate presented mutation at position Asp-94. Moreover, 4 of 35 (11.4%) ciprofloxacin-resistant and one susceptible isolate had no mutation in Ala-90 and Asp-94. No gyrB mutation was observed in all tested M. massiliense isolates. In conclusion, our results have shown that mutations of gyrA codon 90 are frequent and may constitute an important mechanism of resistance to FQ in M. massiliense.