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J Tankovic

Publications and source records attributed to J Tankovic.

At least 19 recordsLinked to original sources
PubMed2004

CdeA of Clostridium difficile, a new multidrug efflux transporter of the MATE family.

The cdeA gene, cloned from Clostridium difficile clinical strain 714 under the control of its natural promoter made Escherichia coli and Clostridium perfringens resistant to ethidium bromide and acriflavin but had no effect on the susceptibility of the hosts to the following antibiotics: norfloxacin, ciprofloxacin, gentamicin, erythromycin, tetracyclin, and chloramphenicol. However, it was responsible for fluoroquinolone resistance in E. coli when it was cloned under the control of the Plac promoter. Quantitative reverse transcriptase (RT)-PCR showed that growth of C. difficile clinical strain 253 in the presence of subinhibitory concentrations of ethidium bromide significantly increased the transcription of cdeA, but this was not observed with ciprofloxacin. The deduced protein was homologous to the protein sequences of known efflux pumps from the third cluster (the so-called DinF branch) of the multidrug and toxic compound extrusion (MATE) family. CdeA caused ethidium bromide energy-dependent efflux in whole cells of E. coli. Efflux activity was stimulated by addition of Na+ ions, suggesting that CdeA, like other pumps of the MATE family, is a Na+-coupled efflux pump. CdeA is the first multidrug efflux transporter identified in C. difficile.

Amino Acid Sequence↗
PubMed2004

Identification of a genetic marker of Helicobacter pylori strains involved in gastric extranodal marginal zone B cell lymphoma of the MALT-type.

BACKGROUND AND AIMS: Gastric extranodal marginal zone B cell lymphoma of the mucosa associated lymphoid tissue (MALT)-type (MZBL) is a rare complication of Helicobacter pylori infection. Currently, no bacterial factor has been associated with the development of this disease. Our aim was to identify genes associated with lymphoma development. METHODS: We used subtractive hybridisation as a tool for comparative genomics between H pylori strains isolated from a patient with gastric MZBL and from a patient with gastritis only. RESULTS: When gastric MZBL strains were compared with gastritis strains, two open reading frames (ORFs) were significantly associated with gastric MZBL: JHP950 (74.4% v 48.7%, respectively; p = 0.023) and JHP1462 (25.6% v 2.6%, respectively; p = 0.004). The prevalence of JHP950 was 48.8% (p = 0.024) in duodenal ulcer strains and 39.3% (p = 0.006) in gastric adenocarcinoma strains, which makes this ORF a specific marker for gastric MZBL strains. In contrast, the prevalence of JHP1462 was 16% (p = 0.545) and 35.7% (p = 0.429) in duodenal ulcer and adenocarcinoma strains, respectively. These ORFs were present in reference strain J99 but not in reference strain 26695. JHP950 is located in the plasticity zone whereas the other, JHP1462, is located outside. Both encode for H pylori putative proteins with unknown functions. CONCLUSION: Despite its low prevalence, the ORF JHP1462 can be considered a candidate marker for H pylori strains involved in severe gastroduodenal diseases. In contrast, the ORF JHP950 has a high prevalence, and is the first candidate marker for strains giving rise to an increased risk of gastric MZBL strains. Further confirmation in other studies is needed.

Adenocarcinoma↗
PubMed2002

Validation of diffusion methods for macrolide susceptibility testing of Helicobacter pylori.

Helicobacter pylori resistance to macrolides is increasing, and the need for susceptibility testing has become crucial. The only standardized method is agar dilution, which is not adapted to clinical practice. The present work aimed: (1) to optimize the technical conditions and to assess the reproducibility of the E-test and disk diffusion method for macrolides susceptibility testing of H. pylori, and (2) to assess the performances of these two phenotypic methods in detecting strains harboring a resistance mechanism to macrolides. We used 191 isolates collected in nine centers of France and Belgium. Phenotypic tests were performed on Mueller-Hinton agar supplemented with 10% horse blood, inoculated with a 2-day-old H. pylori suspension (10(8) CFU/ml), and incubated for 72 hr at 37 degrees C under microaerophilic conditions. The reproducibility studied on two randomly selected strains was better for disk diffusion than for the E-test for both clarithromycin and erythromycin. For a subset of 10 strains, the MICs of erythromycin and clarithromycin did not differ from more than one two-fold dilution when determined by E-test or agar dilution method. The breakpoints were for MICs: 1 mg/L for both clarithromycin and erythromycin and for inhibition diameters, 22 mm for clarithromycin and 17 mm for erythromycin. There was a 100% concordance between susceptibility to erythromycin and clarithromycin. However, the susceptible and resistant populations were better separated by testing erythromycin. Of 34 resistant strains, two lacked the A2142G and A2143G point mutations in 23S rRNA by PCR-RFLP. None of 15 tested sensitive strains were positive for one of these two point mutations. For clinical practice, we recommend to assess macrolide susceptibility of H. pylori by using one of these two phenotypic methods under the described technical conditions.

Anti-Bacterial Agents↗
PubMed2001

Clarithromycin resistance of Helicobacter pylori has a major impact on the efficacy of the omeprazole-amoxicillin-clarithromycin therapy.

Clarithromycin resistance of Helicobacter pylori is relatively frequent in France and is assumed to be the main cause of failure of the proton pump inhibitor-amoxicillin-clarithromycin (PPI-AC) therapy, which is the first-line regimen in our country. We determined the respective effects of clarithromycin primary and secondary resistances on efficacy of the PPI-AC regimen and examined whether failures were associated with persistence of the same strain or with emergence of a new one. Hundred and twenty three H. pylori-infected patients were treated for seven days with omeprazole 20 mg b.d., amoxicillin 1 g b.d., and clarithromycin 500 mg b.d. Eradication was assessed by breath test in 102 patients. MICs of clarithromycin were determined by E-test. Strain genotyping was performed by random amplified polymorphic DNA. The pre-treatment and post-treatment prevalences of clarithromycin resistance were 18.7% (23/123) and 69.2% (9/13), respectively. The rates of eradication were 67.6% (69/102), 78.8% (67/85), and 11.8% (2/17) for all, susceptible and resistant strains, respectively. The post-treatment isolate was available for six patients with a susceptible pre-treatment isolate and a persistent infection; resistance emerged in two patients and was associated with persistence of the pre-treatment strain in one and with selection of a new strain in the other. In conclusion, in our hospital, failures of the PPI-AC therapy are related to both clarithromycin primary and secondary resistances but emergence of secondary resistance does not explain all failures in the initial clarithromycin-susceptible group. In that group a new strain can emerge after failure.

Adolescent↗
PubMed2001

Impact of Helicobacter pylori resistance to clarithromycin on the efficacy of the omeprazole-amoxicillin-clarithromycin therapy.

BACKGROUND: Helicobacter pylori resistance to clarithromycin is relatively frequent in France and is assumed to be the main cause of failure of the proton pump inhibitor-amoxicillin-clarithromycin (proton pump inhibitor-AC) therapy, which is the first-line regimen in France. AIM: To determine the respective effects of clarithromycin primary and secondary resistances on efficacy of the proton pump inhibitor-AC regimen and to determine whether failures are associated with persistence of the same strain or with emergence of a new one. METHODS: A total of 123 H. pylori-infected patients were treated for 7 days with omeprazole 20 mg b.d., amoxicillin 1 g b.d., and clarithromycin 500 mg b.d. Eradication was assessed by breath test in 102 patients. Minimal inhibitory concentrations of clarithromycin were determined by E-test. Strain genotyping was performed by random amplified polymorphic DNA. RESULTS: The pre-treatment and post-treatment prevalences of clarithromycin resistance were 19% (23 out of 123) and 69% (nine out of 13), respectively. The rates of eradication were 68% (69 out of 102), 79% (67 out of 85), and 12% (two out of 17) for all, susceptible and resistant strains, respectively. The post-treatment isolate was available for six patients with a susceptible pre-treatment isolate and a persistent infection. Resistance emerged in two patients and was associated with persistence of the pre-treatment strain in one and with selection of a new strain in the other. CONCLUSIONS: In our hospital, failures of the proton pump inhibitor-AC therapy are related to both clarithromycin primary and secondary resistances, but the emergence of secondary resistance does not explain all of the failures in the initial clarithromycin-susceptible group. In that group a new strain can emerge after failure.

Administration, Oral↗
PubMed2001

Single or double mutational alterations of gyrA associated with fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli.

We looked for the presence of gyrA mutations in seven fluoroquinolone-resistant French clinical isolates of Campylobacter jejuni and Campylobacter coli. Three of the five isolates of C. jejuni and the two isolates of C. coli had high-level resistance to nalidixic acid (MICs 128-256 microg/ml) and ciprofloxacin (MICs 32 microg/ml). A gyrA mutation was found in all these isolates leading to the following substitutions: Thr86-Ile in four cases and Asp90-Tyr for one C. coli strain. One isolate had high-level resistance to nalidixic acid (MIC 64 microg/ml) but low-level resistance to ciprofloxacin (MIC 2 microg/ml) and also carried a gyrA mutation leading to a Thr86-Ala substitution. The last isolate of C. jejuni studied displayed an atypical resistance phenotype: It was resistant to high levels of ciprofloxacin (MIC 64 microg/ml) but remained fully susceptible to nalidixic acid (MIC 2 microg/ml). This phenotype was not explained by the presence of peculiar mutations in gyrA or gyrB. It carried a gyrA mutation leading to a Thr86-Ile substitution and was devoid of gyrB mutation. Despite numerous attempts with various degenerate oligonucleotide primers deduced from conserved regions of known parC genes, we were unable to amplify a corresponding sequence in C. jejuni or C. coli. First-step and second-step in vitro mutants, derived from reference strain C. coli ATCC 33559 with ciprofloxacin or moxifloxacin as selecting agents, were found to carry one and two mutations in gyrA, respectively. In contrast with the results obtained with clinical isolates, a variety of gyrA mutations were obtained in vitro.

Anti-Infective Agents↗
PubMed2000

Evaluation of nitrofurantoin combination therapy of metronidazole-sensitive and -resistant Helicobacter pylori infections in mice.

The main objectives of this study were to determine whether the nitroreductase enzyme encoded by the rdxA gene of Helicobacter pylori was responsible for reductive activation of nitrofurantoin and whether a triple-therapy regimen with nitrofurantoin was able to eradicate metronidazole-sensitive and -resistant H. pylori infections from mice. The susceptibilities to nitrofurantoin of parent and isogenic rdxA mutant strains (three pairs), as well as a series of matched metronidazole-sensitive and -resistant strains isolated from mice (30) and patients (20), were assessed by agar dilution determination of the MIC. Groups of mice colonized with the metronidazole-sensitive H. pylori SS1 strain or a metronidazole-resistant rdxA SS1 mutant were treated with either metronidazole or nitrofurantoin as part of a triple-therapy regimen. One month after the completion of treatment the mice were sacrificed and their stomachs were cultured for H. pylori. The nitrofurantoin MICs for all strains tested were between 0.5 and 4.0 microg/ml. There was no significant difference between the susceptibility to nitrofurantoin of the parental strains and those of respective rdxA mutants or between those of matched metronidazole-sensitive and -resistant H. pylori isolates. The regimen with metronidazole eradicated infection from all eight SS1-infected mice and from one of eight mice inoculated with the rdxA mutant (P < or =0.001). The regimen with nitrofurantoin failed to eradicate infection from any of the six SS1-infected mice (P < or =0.001) and cleared infection from one of seven mice inoculated with the rdxA mutant. These results demonstrate that, despite the good in vitro activity of nitrofurantoin against H. pylori and the lack of cross-resistance between metronidazole and nitrofurantoin, eradication regimens involving nitrofurantoin are unable to eradicate either metronidazole-sensitive or -resistant H. pylori infections from mice.

Animals↗
PubMed2000

Frequent association between alteration of the rdxA gene and metronidazole resistance in French and North African isolates of Helicobacter pylori.

Mutations in the rdxA gene have been associated with the acquisition of resistance to metronidazole in Helicobacter pylori. This gene encodes an NADPH nitroreductase whose expression is necessary for intracellular activation of the drug. We wished to examine whether mutations in rdxA were present in resistant H. pylori isolates infecting either French or North African patients. We determined the complete nucleotide sequences of the rdxA genes from seven French and six North African patients infected with paired resistant and sensitive strains. Genotyping by random amplified polymorphic DNA analysis confirmed the close genetic relatedness of the susceptible and resistant isolates from individual biopsies. Eight French and five North African individual resistant strains were also studied. For the French strains, an alteration in rdxA most probably implicated in resistance was found in 10 cases (seven frameshift mutations, two missense mutations, and one deletion of 211 bp). One to three putative missense mutations were identified in four cases, and a missense mutation possibly not implicated in resistance was discovered in the last case. For the North African strains, an alteration in rdxA was found in eight cases (three frameshift mutations, three missense mutations, one deletion of 6 bp, and one insertion of a variant of IS605). Two strains contained putative missense mutations, and no change was observed in rdxA of the last strain. Thus, inactivation of the rdxA gene is frequently, but not always, associated with resistance to metronidazole in French and North African clinical isolates of H. pylori. In addition, a variety of alterations of rdxA are associated with the resistant phenotype.

Africa, Northern↗
PubMed2000

Roles of gyrA mutations in resistance of clinical isolates and in vitro mutants of Bacteroides fragilis to the new fluoroquinolone trovafloxacin.

We determined whether gyrA mutations were present in fluoroquinolone-resistant laboratory mutants derived from the Bacteroides fragilis reference strain ATCC 25285 and in clinical isolates of B. fragilis. The two first-step mutants selected on ciprofloxacin (CIP) were devoid of gyrA mutations, whereas two of the three CIP-selected second-step mutants studied presented the same gyrA mutation leading to a Ser82Phe change. Unusual GyrA alterations, Asp81Asn or Ala118Val, were detected in two of the three first-step mutants selected on trovafloxacin (TRO), Mt3 and Mt1, respectively. The Ala118Val change had no effect on the susceptibility of Mt1 to CIP. No second-step mutant could be obtained with TRO as a selector. For the 12 clinical isolates studied, a Ser82Phe change in GyrA was found only in the 3 strains which showed the highest levels of TRO resistance (MIC, 4 microgram/ml). Thus, the resistance phenotypes and genotypes observed in fluoroquinolone-resistant clinical isolates of B. fragilis were similar to those found in CIP-selected laboratory mutants, whereas peculiar mutational events could be selected in vitro with TRO.

Anti-Infective Agents↗
PubMed1999

In-vitro activity of moxifloxacin against fluoroquinolone-resistant strains of aerobic gram-negative bacilli and Enterococcus faecalis.

MICs of the new fluoroquinolone, moxifloxacin, and those of ciprofloxacin, ofloxacin and sparfloxacin for 19 genetically characterized fluoroquinolone-resistant strains were determined by the agar dilution method. The MICs of moxifloxacin for Escherichia isolates with one mutation in gyrA (corresponding to Ser83-->Leu or Asp87-->Gly substitution) were 0.25-0.5 mg/L, while those of ciprofloxacin, ofloxacin and sparfloxacin were 0.06-0.25, 1 and 0.12-0.5 mg/L, respectively. These values were four- to 16-fold higher than those of the same antibiotics for the wild-type strain, E. coli KL16. Similar results were observed with clinical isolates of Salmonella spp. harbouring one mutation in gyrA leading to the substitution of Ser83 by Phe or Tyr. In the presence of two mutations in the E. coli gyrA gene, the MICs of moxifloxacin ciprofloxacin, ofloxacin and sparfloxacin were 2, 0.5, 4 and 1 mg/L, respectively; these were 32 times higher than the MICs of these agents for E. coli KL16. The MICs of the four quinolones for triple mutants with two mutations in gyrA and one in parC were even higher, i.e. 8, 8, 16 and 8-16 mg/L, respectively. The MICs of moxifloxacin for Campylobacter coli and Campylobacter jejuni strains with a gyrA mutation leading to Thr86-->Ile substitution ranged from 1 to 2 mg/L, while the MICs of ciprofloxacin, ofloxacin and sparfloxacin were 16-32 mg/L, 8-16 and 4-8 mg/L, respectively. For high-level ciprofloxacin-resistant (MICs of 32 mg/L) clinical isolates of Enterococcus faecalis with one substitution at position 83 in GyrA (E. coli coordinates), the MICs of moxifloxacin, ofloxacin and sparfloxacin were 8-16, > or = 128 and 32 mg/L respectively. In conclusion, moxifloxacin and other fluoroquinolones exhibit cross-resistance against aerobic gram-negative bacilli and enterococci. The in-vitro activity of moxifloxacin was greater than that of ofloxacin and slightly less than that of ciprofloxacin and sparfloxacin against Enterobacteriaceae, but greater than those of the three other compounds tested against Campylobacter spp and E. faecalis.

Anti-Infective Agents↗
PubMed1998

Co-detection of Helicobacter pylori and of its resistance to clarithromycin by PCR.

Our aim was to develop a rapid molecular test based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and making it possible to detect Helicobacter pylori directly from gastric biopsy samples, and to test its susceptibility to clarithromycin. A 629-bp fragment of the 23S rRNA gene of H. pylori was amplified by PCR and the mutations responsible for clarithromycin resistance were detected with Bsa1 and Bbs1 restriction endonucleases. Thirty-five gastric samples were tested in parallel by standard microbiologic methods (culture and clarithromycin susceptibility testing with E-test strips) and by PCR-RFLP. The 10 culture-negative samples were also PCR-negative. Sixteen out of the 25 culture-positive samples (64%) were PCR-positive. RFLP analysis could be done in 12 cases and the results were in agreement with those of the E-test: susceptibility in five cases, resistance in seven (six A2144G mutations and one A2143G mutation).

Anti-Bacterial Agents↗
PubMed1998

Induction of nitric oxide synthase in vivo and cell injury in rat duodenal epithelium by a water soluble extract of Helicobacter pylori.

1. Helicobacter pylori (Hp) infection, which involves the gastric antrum and duodenal mucosa, may be involved in peptic ulceration by stimulating the local release of cytoxic or pro-inflammatory factors. 2. Nitric oxide (NO) is known to be cytotoxic at high concentration. The aim of the present study was therefore to investigate the ability of a water soluble extract of Hp to induce NO synthase in duodenal mucosa and epithelial cells following its administration in vivo in rats and determine its association with cell damage. 3. Administration of Hp water extract (4 ml kg(-1)) led to the expression of the calcium-independent inducible nitric oxide synthase (iNOS) after 4 h in the duodenum, determined as [14C]-arginine conversion to citrulline. 4. This iNOS activity was not reduced by pretreatment with anti-neutrophil serum (0.4 ml kg(-1), i.p., 3 h before challenge). However, dexamethasone pretreatment (1 mg kg(-1), i.v., 2 h before the extract), or administration of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 5 mg kg(-1), i.v., 2.5 h after the extract) reduced this activity. 5. Furthermore, iNOS was expressed in duodenal isolated epithelial cells 4 h after the i.v. challenge with the extract, at a time when the cellular viability was also reduced, as assessed by trypan blue exclusion. 6. Dexamethasone pretreatment, administration of L-NAME, or pretreatment with polymyxin B (1 mg kg(-1), i.v.) which binds endotoxin, reduced both the iNOS activity and epithelial cell damage. 7. The induction of NO synthase by the Hp extract thus results in duodenal epithelial cell injury and such actions could play a role in pathogenesis of peptic ulcer disease.

Animals↗
PubMed1998

Analysis of the mutations involved in fluoroquinolone resistance of in vivo and in vitro mutants of Escherichia coli.

We searched for the mutations involved in high-level fluoroquinolone resistance (ciprofloxacin MIC > or = 8 microg/ml) of 11 clinical isolates of Escherichia coli. trans-Complementation tests with the wild-type gyrA and parC genes were positive for all strains whereas negative results were observed with the wild-type gyrB and parE genes. By PCR and sequencing, two mutations in gyrA, leading to Ser-83 --> Leu and Asp-87 --> Asn (7) or Gly (2) or Tyr (1) changes, were found in 10 strains, the eleventh presenting only the Ser-83 --> Leu change. In addition, all strains carried one change in ParC: Ser-80 --> Ile (8) or Arg (2); Glu-84 --> Lys (1). We described a novel and simple method permitting detection of the mutations in parC at codon 80, PCR-RFLP with HaeII. In vitro mutants, selected with ciprofloxacin in three successive steps were also studied. The first-step mutants were complemented by pJSW101 (gyrA+) but not by pEN260 (parC+), whereas the second-step and third-step mutants were complemented by both plasmids. Mutations occurred in the following order: (i) gyrA at codon 83 (Ser to Leu change), (ii) parC at codon 80 (Ser to Ile change), and (iii) gyrA at codon 87 (Asp to Asn change). Thus, these sequential mutations appear to be frequently involved in high-level fluoroquinolone resistance of E. coli.

Anti-Infective Agents↗
PubMed1998

[Comparison of quantifying Helicobacter pylori gastric infection by culture, histology and C13 urea breath test].

OBJECTIVES: In Helicobacter pylori infection, the bacterial burden may play a role in the pathogenesis of gastric or duodenal ulcerated lesions. It could also influence the results of antimicrobial therapy. No simple test has been validated to quantify Helicobacter pylori density. The aim of this study was to determine the value of histology and/or 13C-urea breath test to quantify the infection as compared with quantitative culture, taken as a reference method. PATIENTS AND METHODS: Biopsies samples were taken from the antrum at endoscopy in 72 patients. Thirty-seven patients with positive urease test at 20 minutes were enrolled in the study. Bacterial density was evaluated from biopsies by quantitative culture and semi-quantitative histological examination (score from 0 to 3). The bacterial density was evaluated as well by 13C-urea breath test from the proportion of 13CO2 in exhaled air (delta 13CO2) at 20, 40, and 60 minutes as compared with the basal level. RESULTS: The bacterial density, evaluated by quantitative culture ranged from 5 CFU to 110,000 CFU per mg of tissue. By histology, a score 1 was found in 5 patients, a score 2 in 17, and a score 3 in 15. delta of 13CO2 measured by 13C-urea breath test ranged from 0.2 to 117.5, from 0.2 to 102, and from 0.6 to 66.7 at 20, 40 and 60 minutes respectively. The quantity of bacteria measured by culture was not significantly higher for these with a score of 3 as compared with those with a pooled score of 1 and 2 (P < 0.05). No significant correlation was found between the results of quantitative culture and these of breath test. CONCLUSION: In practice, evaluation of bacterial burden by a histological score seems only accurate for the most severe density (score 3). The 13C-urea breath test does not allow a reliable quantitative evaluation.

Adult↗
PubMed1997

Quinolone resistance locus nfxD of Escherichia coli is a mutant allele of the parE gene encoding a subunit of topoisomerase IV.

The locus nfxD, which contributes to high-level quinolone resistance in Escherichia coli KF111b (gyrAr nfxB nfxD), is only expressed in the presence of a gyrA mutation, and maps to the region of the parC and parE genes, was outcrossed into strain KF130, creating strain DH161 (gyrAr nfxD). DNA sequence analysis of DH161 revealed no changes in the topoisomerase IV parC quinolone resistance-determining region but did identify a single T-to-A mutation in parE at codon 445, leading to a change from Leu to His. Full-length cloned parE+ partially complemented the resistance phenotype in KF111b and DH161, but did not complement the resistance phenotype in strain KF130 (gyrAr). No complementation was seen with cloned, truncated parE+. To confirm these findings, gyrAr was first outcrossed from KF130 into E. coli W3110parE10 [parE temperature sensitive(Ts)] and KL16. The transduced strains KL16 and W3110parE10 were subsequently transformed with plasmids containing cloned parE from DH161 or KL16. Cloned parE from DH161 increased norfloxacin resistance in the parE(Ts) background twofold at 30 degrees C and fourfold at 42 degrees C compared to those for cloned parE from KL16. The same experiment with a non-Ts background revealed a twofold increase in the norfloxacin MIC at both 30 and 42 degrees C. These data identify the nfxD conditional resistance locus as a mutant allele of parE. This report is the first of a quinolone-resistant parE mutant and confirms the role of topoisomerase IV as a secondary target of norfloxacin in E. coli.

Alleles↗