Prevalence of Primary Fluoroquinolone Resistance Among Clinical Isolates of Helicobacter pylori at a University Hospital in Southern Taiwan

Background:  Fluoroquinolone-containing therapy is effective in eradicating Helicobacter pylori . However, the resistance rate of H. pylori to fluoroquinolones in Taiwan has not yet been reported. In this study, we aimed to investigate the susceptibility to antibiotics commonly used in eradication schedules and fluoroquinolones in H. pylori .
Methods:  A total of 210 clinical isolates of H. pylori were collected from April 1998 to September 2007 from patients in southern Taiwan. The in vitro activities of six antimicrobial agents were determined by the agar dilution method and Etest. The mutations in quinolone resistance-determining regions of gyrA and gyrB were investigated by direct sequencing.
Results:  Overall, 5.7% of the isolates were resistant to ciprofloxacin and levofloxacin. The resistance rate to amoxicillin, clarithromycin, metronidazole, and tetracycline was 1.0% (two of 210), 9.5% (20 of 210), 27.6% (58 of 210), and 0.5% (one of 210), respectively. The resistance rate to either ciprofloxacin or to levofloxacin increased from 2.8% (1998–2003) to 11.8% (2004–2007). The mutations in gyrA at N87 or D91 had an impact on primary fluoroquinolone resistance in H. pylori . Garenoxacin, but not moxifloxacin, had a good in vitro inhibitory effect against ciprofloxacin/levofloxacin-resistant strains compared with objective minimal inhibitory concentration values.
Conclusions:  Drug resistance to ciprofloxacin and levofloxacin in H. pylori collected from 2004 to 2007 increased significantly compared with resistance level observed during 1998–2003. The continuous surveillance of quinolone resistance among H. pylori is important in this area.     

Prevalence and mechanisms of resistance to fluoroquinolones in Helicobacter pylori strains from patients living in Belgium

BACKGROUND: Because of the increasing resistance of Helicobacter pylori against metronidazole and clarithromycin, alternative regimens including newer fluoroquinolones have been developed. We aimed to assess the prevalence as well as the mechanisms of this resistance in clinical isolates originating from patients living in Belgium. METHODS: Minimal inhibitory concentration (MIC) values of ciprofloxacin, levofloxacin, and moxifloxacin were determined by Etest method on 488 H. pylori isolates originating from patients who underwent upper gastrointestinal endoscopy at 10 different centers. Resistant strains (MIC values > 1 microg/ml) were evaluated for the presence of point mutations in the quinolone resistance-determining region (QRDR) of the gyrA by amplification and nucleotide sequence. RESULTS: Eighty-two (16.8%) of the strains were found resistant to all fluoroquinolones and 70 of these were further analyzed. Homogeneous and heterogeneous resistance were observed in 55 (78.6%) and in 15 (21.4%) of the strains, respectively. QRDR sequencing revealed various mutations of the codons corresponding to Asn-87 and Asp-91 in all isolates with homogeneous resistance. However, in 12 of 15 strains displaying heterogenous resistance, mutations were only detected after subcultures of isolated colonies growing within the ellipse inhibition zone of the E-test. Amino acid substitutions in the QRDR of GyrA could not be directly related with the MIC values of the isolates. Fluoroquinolone-resistant mutants were easily selected in vitro at frequencies ranging between 10(-6) and 10(-7). Such selected mutants stably persisted after several serial passage in antibiotic-free agar. CONCLUSIONS: These results suggest that H. pylori resistance to fluoroquinolones is occurring at a high frequency in the Belgian population and that it is essentially mediated through a variety of point mutations occurring in a few loci of GyrA. As a consequence, we strongly suggest to determine the susceptibility of the infecting isolates to fluoroquinolones before administration of an anti-H. pylori regimen including these agents.     

Fluoroquinolone resistance in Helicobacter pylori: role of mutations at position 87 and 91 of GyrA on the level of resistance and identification of a resistance conferring mutation in GyrB

Background and Aims: Fluoroquinolone‐containing regimens have been suggested as an alternate to standard triple therapy for the treatment of Helicobacter pylori infections. To determine the relationship between fluoroquinolone resistance and mutations of GyrA and GyrB in H. pylori, we exchanged the mutations at positions 87and 91 of GyrA among fluoroquinolone‐resistant clinical isolates. GyrB of a strain with no mutations in GyrA was also analyzed to identify mechanisms of resistance to norfloxacin. Materials & Methods: Natural transformation was performed using the amplified fragment of the gyrA and gyrB gene as donor DNA. The amino acid sequences of GyrA and GyrB were determined by DNA sequencing of the gyrA and gyrB genes. Results: Norfloxacin‐resistant strains which had mutations at position 87 and 91 became susceptible when the mutations were converted to the wild type. When the mutation from Asp to Asn at position 91 was exchanged to the mutation from Asn to Lys at position 87, the MIC to levofloxacin, gatifloxacin, and sitafloxacin increased. Norfloxacin‐resistant strain TS132 with no mutations in GyrA but had a mutation at position 463 in GyrB. Transformants obtained by natural transformation using gyrB DNA of TS132 had a mutation at position 463 of GyrB and revealed resistant to norfloxacin and levofloxacin. Conclusion: Mutation from Asn to Lys at position 87 of GyrA confers higher resistance to levofloxacin and gatifloxacin than does mutation from Asp to Asn at position 91. We propose that mutation at position 463 in GyrB as a novel mechanism of fluoroquinolone resistance in H. pylori.     

Real-time PCR detection of Pseudomonas aeruginosa in clinical and municipal wastewater and genotyping of the ciprofloxacin-resistant isolates

Real-time quantification of Pseudomonas aeruginosa was performed in various wastewater systems including clinical, municipal wastewaters and inflow from a wastewater treatment plant. The highest concentrations of P. aeruginosa-specific targets were detected in clinical wastewaters. Limitations of the detection system resulting from inhibition or cross-reaction were identified. Ciprofloxacin-resistant P. aeruginosa strains were isolated after specific enrichment from clinical and municipal wastewaters. In some cases they were also cultivated from effluent of a wastewater treatment plant, and from its downstream river water. A total of 119 isolates were phenotypically characterized as ciprofloxacin-resistant via antibiogram testing. Subsequently, the fluoroquinolone-resistance-mediating mutations in the genes gyrA codon positions 83 and 87, gyrB codon position 466 and parC codon positions 87 and 91 were determined by mini-sequencing. Ciprofloxacin resistance was mainly associated with mutations in gyrA codon position 83 and parC mutation in codon positions 87 or 91 of the bacterial gyrase and topoisomerase II genes. All ciprofloxacin-resistant P. aeruginosa strains were compared with genotypes from clinical data of fluoroquinolone-resistant P. aeruginosa infections. The results were in agreement with data from clinical analyses, with the exception that no gyrA 87 and no gyrB mutations were found in ciprofloxacin-resistant P. aeruginosa wastewater isolates.     

Rapid method for detection of gyrA and grlA mutations in unrelated strains of Staphylococci susceptible and resistant to levofloxacin.

A panel of 150 clinical isolates of methicillin resistant and susceptible Staphylococci were investigated using a rapid and simple PCR-RFLPs technique to detect DNA nucleotide changes at the site of the most frequently reported mutations in grlA (codons 79, 80) and gyrA (codons 83, 84) genes which confer fluorquinolone resistance in Staphylococci. Convergent dual mutations in and gyrA and grlA were found in all strains exhibiting resistance to ciprofloxacin (MIC, 8 to > or =128 mg/l) and levofloxacin (MIC, 8 to > or =64 mg/l). Mutations in grlA and gyrA were also found in strains susceptible to levofloxacin and resistant to ciprofloxacin. In our sample no strains with only grlA mutations were found. Our data indicate that methicillin-resistant fluorquinolone-resistant strains are likely to have mutations in both grlA and gyrA. In contrast, methicillin-susceptible strains do not show any mutation. The genetic relatedness of a sample of representative epidemiologically unrelated MRSA strains, tested by PFGE and rep-PCR, are in agreement with the hypothesis of a clonal selection of these resistant strains.     

Rapid detection of gyrA and parC mutations in fluoroquinolone-resistant Neisseria gonorrhoeae by denaturing high-performance liquid chromatography

The detection of DNA sequence variation is fundamental to the identification of the genomic basis of phenotypic variability. Denaturing high-performance liquid chromatography (DHPLC) is a novel technique that is used to detect mutations in human DNA. This is the first report that this technique is used as a tool to detect mutations in genes encoding fluoroquinolone resistance in Neisseria gonorrhoeae. Eighty-one strains of N. gonorrhoeae were used in this study. Genomic DNA from each strain was subjected to PCR amplification of 225 bp in gyrA and 166 bp in parC spanning the fluoroquinolone-resistance determining regions (QRDRs). After we performed DNA sequencing of these amplicons and identification of mutations in the QRDRs, DHPLC was undertaken to investigate whether its results correlate the distinctive chromatogram with their DNA mutations pattern. The profilings detected by DHPLC completely corresponded to the results of the DNA sequencing in mutation patters in gyrA and parC genes. They resulted in the following amino acid substitutions: Ser-91Phe, Asp-95Gly, and Asp-95Asn in gyrA; and Gly-85Asp, Asp-86Asn, Ser-87Arg, and Ser-88Pro in parC, respectively. These mutations existed alone or as combinations, and we identified five mutations patterns in gyrA and six in parC including wild-type. These mutations and their patterns could be rapidly and reproducibly identified from the PCR products using DHPLC, producing specific peak patterns that correlate with genotypes. This novel detection system facilitates the detection of resistance alleles, providing a rapid (5 min per sample), economic (96 sample per run), and reliable technique for characterizing fluoroquinolone resistance in N. gonorrhoeae.     

Primary antibiotic resistance of Helicobacter pylori isolated from Beijing children

Background: The antimicrobials resistance of Helicobacter pylori (H. pylori) was able to sharply decline the eradication rate of H. pylori both in adults and children, but there are limited studies about the primary antibiotic resistance and the related gene mutations, specifically in China. Materials and Methods: The primary resistance to 9 antibiotics of 73 H. pylori strains isolated from gastric biopsies of children recruited at Beijing Children’s Hospital was assessed, and the mutations in 23S rRNA gene of 65 macrolide‐resistant strains and in gyrA and gyrB of 12 quinolone‐resistant strains were investigated. Results: The resistance rate to clarithromycin, azithromycin, metronidazole, levofloxacin, moxifloxacin, and rifampicin was 84.9%, 87.7%, 61.6%, 13.7%, 15.1%, and 6.8%, respectively. No resistance to amoxicillin, gentamicin, and tetracycline was observed. Dual, triple, and quadruple antibacterial resistant percentage was 46.6% (34/73), 15.1% (11/73), and 2.7% (2/73), respectively. The gene mutation rate of A2142C, A2142G, and A2143G in 23S rRNA gene was 1.5% (1/65), 6.2% (4/65), and 84.6% (55/65), respectively. The detection rate of mutations of Asn87, Asp91, and Met191 in GyrA was 41.7% (5/12), 25% (3/12), and 25% (3/12), respectively. Conclusion: The high prevalence of primary antibiotic resistance was out of expectation in H. pylori strains isolated from the children in Beijing. Antibiotic susceptibility should be made clear before the antibiotic was used in the anti‐H. pylori therapy in this population. The A2143G was the most populated mutation in macrolide‐resistant strains, and Asn87 and Asp91 of GyrA were the most common mutation points in quinolone resistance strains.     

New insights into fluoroquinolone resistance in Mycobacterium tuberculosis: functional genetic analysis of gyrA and gyrB mutations

Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis.     

Gemifloxacin can partially overcome quinolone resistance of H. pylori with gyrA mutation in Taiwan

Backgrounds: 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. Materials and Methods: 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. Results: 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). Conclusion: Gemifloxacin is superior to levofloxacin in antimicrobial activity against clinical H. pylori isolates, and even overcome some levofloxacin resistance.     

Evaluation of a high-density oligonucleotide array for characterization of grlA, grlB, gyrA and gyrB mutations in fluoroquinolone resistant Staphylococcus aureus isolates

Using high-density oligonucleotide array technology, 30 Staphylococcus aureus strains were studied for the presence of mutations in genes involved in fluoroquinolone resistance: grlA, gyrA, grlB and gyrB. For the two most important genes, gyrA and grlA, correlation with sequencing reached 95.1%. If all genes were considered, correlation was 88.8%.     

Quinolone action in Escherichia coli cells carrying gyrA and gyrB mutations

Abstract We have isolated spontaneous mutant strains of Escherichia coli KL16 showing different levels of nalidixic acid (NAL) resistance. From 40 independent mutants, 36 had gyrA and four had gyrB mutations. Most of the gyrA mutations (30/36) conferred high level NAL resistance. In contrast, the only gyrB mutation that conferred a relatively high level of NAL resistance also determined enhanced susceptibility to quinolones with a piperazinyl substituent at C7 position of the quinolone ring (amphoteric quinolones). This gyrB mutation (denoted gyrB1604 ), jointly with a gyrA mutation (denoted gyrA972 ) which confers a high level of quinolone resistance, were used to construct strain IC2476, carrying the two gyr mutant alleles. The susceptibility of this strain to amphoteric quinolones (pipemidic acid, norfloxacin and ciprofloxacin) was similar to that of the gyrA972 single mutant. This result indicates that the change in GyrA subunit which determines a high level of quinolone-resistance has the capacity to mask the hypersusceptibility to amphoteric quinolones promoted by the GyrB1604 mutant subunit. This capacity was further confirmed by studying the effects of ciprofloxacin (CFX) on gyrase inhibition in the gyrA972 gyrB1604 strain.     

Genetical conditioning of fluoroquinolone resistance mechanisms of clinical enterococcus faecalis strains. II. The mutations present in the qrdrs of gyrA, gyrB, parC and parE genes

The analyze selected fluoroquinolone resistance mechanisms of clinical E. faecalis strains was presented. In the second part of the study of genetic polymorphisms and mutations in the QRDRs of gyrA, gyrB, parC and parE genes were analyzed. The MSSCP technique and DNA sequencing were used. The activity (MICs) of ciprofloxacin, sparfloxacin and moxifloxacin were determined against 180 tested strains. The MSSCP method allows rapid screening of the genetic polymorphisms analyze of gyrA, gyrB, parC i parE genes. The amino acid substitutions of GyrA, GyrB and ParC were observed. The results indicate that mutations present among clinical E. faecalis strains associated with high level resistance to fluoroquinolons.     

7-day triple therapy of Helicobacter pylori infection with levofloxacin, amoxicillin, and high-dose esomeprazole in patients with known antimicrobial sensitivity

BACKGROUND AND AIMS: Failed primary anti-Helicobacter pylori therapy results in a high rate of antimicrobial resistance. This necessitates a search for new regimens to cure H. pylori infection. The aim of this study was to evaluate the efficacy and tolerability of a new levofloxacin-containing 7-day triple therapy and to compare it with that of standard French triple therapy in patients with known H. pylori susceptibility to MET (metronidazole) and CLA (clarithromycin). PATIENTS AND METHODS: Sixty-one patients with documented antibiotic sensitivity (E-test) and an indication for anti-H. pylori treatment based on the Maastricht Consensus 2/2000 guidelines were randomized to receive either esomeprazole 2 x 40 mg, levofloxacin 2 x 500 mg, and amoxicillin 2 x 1 g for 7 days (ELA, n = 30), or esomeprazole 2 x 20 mg, clarithromycin 2 x 500 mg, and amoxicillin 2 x 1 g for 7 days (ECA, n = 31). A cure check was performed 4-6 weeks after conclusion of therapy. RESULTS: Sixty-one patients were randomized to the two treatment groups. Twenty-eight of 30 patients of the ELA group were available for per-protocol (PP) analysis, of whom 26 (92.9% CI: 76-99%; intention-to-treat [ITT] analysis 86.7% CI: 68-96%) became H. pylori negative compared with 26 of the 31 patients of the ECA group (83.9%, CI: 66-93% both PP and ITT analyses). Five patients of the ELA group showed CLA resistance, three of whom also showed MET resistance, and all five were treated successfully. Two patients with levofloxacin-resistant strains, one in each group, were cured. Both regimens were generally well tolerated with minor adverse events being seen in 15 patients (51.7%) of the ELA group and in 13 (40.6%) of the ECA group. None of the patients discontinued treatment prematurely due to adverse events. CONCLUSION: The data of this pilot study suggest a better than 80% efficacy of the new 7-day levofloxacin triple therapy, which is within the range of the French triple therapy in patients with MET- and CLA-susceptible strains. The data suggest that the new levofloxacin triple therapy may also be an option in patients with MET- and CLA-resistant H. pylori strains.     

Mutants of Escherichia coli defective for replicative transposition of bacteriophage Mu.

We isolated 142 Hir- (host inhibition of replication) mutants of an Escherichia coli K-12 Mu cts Kil- lysogen that survived heat induction and the killing effect of Mu replicative transposition. All the 86 mutations induced by insertion of Tn5 or a kanamycin-resistant derivative of Tn10 and approximately one-third of the spontaneous mutations were found by P1 transduction to be linked to either zdh-201::Tn10 or Tn10-1230, indicating their location in or near himA or hip, respectively. For a representative group of these mutations, complementation by a plasmid carrying the himA+ gene or by a lambda hip+ transducing phage confirmed their identification as himA or hip mutations, respectively. Some of the remaining spontaneously occurring mutations were located in gyrA or gyrB, the genes encoding DNA gyrase. Mutations in gyrA were identified by P1 linkage to zei::Tn10 and a Nalr gyrA allele; those in gyrB were defined by linkage to tna::Tn10 and to a gyrB(Ts) allele. In strains carrying these gyrA or gyrB mutations, pBR322 plasmid DNA exhibited altered levels of supercoiling. The extent of growth of Mu cts differed in the various gyrase mutants tested. Phage production in one gyrA mutant was severely reduced, but it was only delayed and slightly reduced in other gyrA and gyrB mutants. In contrast, growth of a Kil- Mu was greatly reduced in all gyrase mutant hosts tested.     

Screening method for Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones by PCR-restriction fragment length polymorphism

Salmonella enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones (MICs of ciprofloxacin, 0.25 to 2 microg/ml) have a mutation at codon either Ser-83 or Asp-87 of gyrA gene. A screening method by PCR-restriction fragment length polymorphism (PCR-RFLP) was designed to screen the mutations at codon Ser-83 and Asp-87 of the gyrA gene of S. enterica serovar Typhi and serovar Paratyphi A clinical isolates. This method successfully screened the gyrA mutations of S. enterica serovar Typhi and serovar Paratyphi A with reduced susceptibility to fluoroquinolones.     

Identification of the tip-encoded receptor in bacterial sensing.

Relaxation of titratable supercoils in bacterial nucleoids was measured following treatment of topA mutants with coumermycin or oxolinic acid, inhibitors of DNA gyrase. Relaxation occurred after treatment of the mutants with either inhibitor. We detected no significant difference in relaxation between topA- and topA+ strains treated with coumermycin. This finding, together with previous observations, supports the idea that relaxation caused by coumermycin probably arises from the relaxing activity of gyrase itself. The source of DNA relaxation caused by oxolinic acid was not identified. Nucleoid supercoiling can be increased by adding oxolinic acid to a strain that carries three topoisomerase mutations: delta topA, gyrB225, and gyrA (Nalr) (S. H. Manes, G. J. Pruss, and K. Drlica, J. Bacteriol. 155:420-423, 1983). We found that this increase in supercoiling requires partial sensitivity to the drug and at the delta topA and gyrA mutations. Full resistance to oxolinic acid in the presence of the delta topA, gyrB225, and gyrA mutations was conferred by an additional mutation that maps at or near gyrB.     

Increased production of a knotted form of plasmid pBR322 DNA in Escherichia coli DNA topoisomerase mutants

Plasmid pBR322 prepared from Escherichia coli strains carrying deletion of the DNA topoisomerase I gene (delta topA) with a compensatory mutation of the DNA gyrase gene (gyrA or gyrB) and from their TopA+ transductants was analyzed by agarose gel electrophoresis followed by electron microscopy, and compared with that from isogenic wild-type strains. It was found that about 1% of the plasmid DNA molecules was a knotted species in the topA+ gyr+ strains W3110 and DM4100, while strains DM750 (delta topA gyrA224), DM800 (delta topA gyrB225), SD275 (topA+ gyrA224) and SD108 (topA+ gyrB225) produced six to ten times as much knotted DNA as the topA+ gyr+ controls. The results suggest that the increased production of knotted pBR322 DNA is closely related to mutations of the gyrase genes.     

Mutations of Helicobacter pylori associated with fluoroquinolone resistance in Korea

Background and Aim: Fluoroquinolone resistance of Helicobacter pylori is known to be dependent on mutations in the QRDR of gyrA. This study was performed to investigate the distribution of gyrA point mutations and to evaluate the impact of the mutations on second‐line H. pylori eradication therapy. Methods: After H. pylori isolation from gastric mucosal specimens, fluoroquinolone resistance was examined using the agar dilution method. DNA sequencing of the QRDR of gyrA was performed in 89 fluoroquinolone‐resistant and 27 fluoroquinolone‐susceptible isolates. Transformation experiments were performed to confirm mutations in the resistant strains. The eradication rates of moxifloxacin‐containing triple therapy were evaluated depending on the resistance of fluoroquinolone. Results: The gyrA mutations were detected in 75.3% (55 of 73 strains) of the primary resistant strains and 100% (16 strains) of the secondary resistant strains. The most common mutations were Asp‐91 (36.0%) and Asn‐87 (33.7%). The MIC values in the transformed strains differed depending on the gyrA mutations, N87, and D91. Six patients with fluoroquinolone‐resistant strains received moxifloxacin‐containing triple therapy as the second‐line therapy, and two of three patients with Asn‐87 mutations (66.7%) failed in the eradication. By contrast, three patients with Asp‐91 mutations had successful eradication treatment. Conclusions: Fluoroquinolone resistance of H. pylori was caused by gyrA Asn‐87 and Asp‐91 point mutations. The Asn‐87 mutation seems to be an important determinant of failure of fluoroquinolone‐containing triple eradication therapy based on eradication results.     

Primary antibiotic resistance and associated mechanisms in Helicobacter pylori isolates from Senegalese patients

Background

Antibiotic combination therapy for Helicobacter pylori eradication must be adapted to local resistance patterns, but the epidemiology of H. pylori resistance to antibiotics is poorly documented in Africa. The aim was to determine the antibiotic resistance rates, as well as the associated molecular mechanisms, of strains isolated in Dakar, Senegal.

Methods

One hundred and eight H. pylori strains were isolated between 2007 and 2009 from 108 patients presenting with upper abdominal pain to the Gastroenterology Department of Le Dantec Hospital. Antimicrobial susceptibility testing was performed for amoxicillin, clarithromycin, metronidazole, levofloxacin and tetracyclin using the E-test method. Mutations in the 23S rRNA gene of clarithromycin-resistant strains and in gyrA and gyrB of levofloxacin-resistant strains were investigated.

Results

Isolates were characterized by no resistance to amoxicillin (0%), tetracycline (0%), and very low rate of resistance to clarithromycin (1%), but a high rate of resistance to metronidazole (85%). The clarithromycin-resistant strain displayed the A2143G mutation. A worrying rate of levofloxacin resistance was detected (15%). N87I and D91N were the most common mutations in the quinolone-resistance-determining region of gyrA.

Conclusions

The first-line empirical regimen for H. pylori eradication in Senegal should include clarithromycin. Increasing rates of fluoroquinolone resistance detected should discourage the use of levofloxacin-containing regimens without prior antimicrobial susceptibility testing.     

Frequency and Geographic Distribution of gyrA and gyrB Mutations Associated with Fluoroquinolone Resistance in Clinical Mycobacterium Tuberculosis Isolates: A Systematic Review

Background

The detection of mutations in the gyrA and gyrB genes in the Mycobacterium tuberculosis genome that have been demonstrated to confer phenotypic resistance to fluoroquinolones is the most promising technology for rapid diagnosis of fluoroquinolone resistance.

Methods

In order to characterize the diversity and frequency of gyrA and gyrB mutations and to describe the global distribution of these mutations, we conducted a systematic review, from May 1996 to April 2013, of all published studies evaluating Mycobacterium tuberculosis mutations associated with resistance to fluoroquinolones. The overall goal of the study was to determine the potential utility and reliability of these mutations as diagnostic markers to detect phenotypic fluoroquinolone resistance in Mycobacterium tuberculosis and to describe their geographic distribution.

Results

Forty-six studies, covering four continents and 18 countries, provided mutation data for 3,846 unique clinical isolates with phenotypic resistance profiles to fluoroquinolones. The gyrA mutations occurring most frequently in fluoroquinolone-resistant isolates, ranged from 21–32% for D94G and 13–20% for A90V, by drug. Eighty seven percent of all strains that were phenotypically resistant to moxifloxacin and 83% of ofloxacin resistant isolates contained mutations in gyrA. Additionally we found that 83% and 80% of moxifloxacin and ofloxacin resistant strains respectively, were observed to have mutations in the gyrA codons interrogated by the existing MTBDRsl line probe assay. In China and Russia, 83% and 84% of fluoroquinolone resistant strains respectively, were observed to have gyrA mutations in the gene regions covered by the MTBDRsl assay.

Conclusions

Molecular diagnostics, specifically the Genotype MTBDRsl assay, focusing on codons 88–94 should have moderate to high sensitivity in most countries. While we did observe geographic differences in the frequencies of single gyrA mutations across countries, molecular diagnostics based on detection of all gyrA mutations demonstrated to confer resistance should have broad and global utility.