Helicobacter pylori acquisition of metronidazole resistance by natural transformation in vitro

AIM To study whether Helicobacter pylori is naturally transformable. METHODS Transformation was performed in BHI broth supplemented with horse serum and yeast extract. Genomic DNA extracted from a metronidazole resistant H. pylori strain was added to H. pylori broth culture. The mixture was incubated at microaerophilic atmosphere. The DNA treated cells were plated on blood agar containing 8mg/L metronidazole to select for transformants. Sterile distilled water was used as a negative DNA control. The DNA profiles of transformants were compared with that of their parent strains by randomly amplified polymorphic DNA (RAPD) fingerprinting. RESULTS Transformation of H. pylori with DNA from a metronidazole resistant strain as a marker was demonstrated. Out of the 12 strains of H. pylori tested, 9 (75%) strains were found to be transformable. The transformation frequencies ranged from 3 4×10 -6 to 2 4×10 -4 . By RAPD, DNA fingerprints of the transformants and their parent strains showed no change in DNA profiles though transformants were all resistant to metronidazole as compared with their metronidazole sensitive parent strains. CONCLUSION Helicobacter pylori is naturally transformable which might be one of the ways that H. pylori develops resistance to metronidazole.

Prevalence of primary Helicobacter pylori resistance to metronidazole and clarithromycin in Singapore

INTRODUCTIONEradication of Helicobacter pylori,a bacteriumresiding in stomach and causing peptic ulcer disease,can be achieved by using combination therapiesconsisting of one or two antibiotics with a protonpump inhibitor (PPI).The major antibiotics widelyused in the regimens to eradicate H.pylori aremetronidazole and clarithromycin.However,resistance to these antibiotics by H.pylori

Aspirin increases susceptibility of Helicobacter pylori to metronidazole by augmenting endocellular concentrations of antimicrobials

AIM： To investigate the increasing the susceptibility pylon） to metronidazole. mechanisms of aspirin of Helicobacter pylori （H METHODS： Hpylori reference strain 26695 and two metronidazole-resistant isolates of H pylori were included in this study. Strains were incubated in Brucella broth with or without aspirin （1 mmol/L）. The rdxA gene of Hpylori was amplified by PCR and sequenced. The permeability of Hpylori to antimicrobials was determined by analyzing the endocellular radioactivity of the cells after incubated with [7-^3H]-tetracycline. The outer membrane proteins （OMPs） of Hpylori 26695 were depurated and analyzed by SDS-PAGE. The expression of 5 porins （hopA, hopB, hopC, hopD and hopE） and the putative RND efflux system （hefABC） of H pylori were analyzed using real-time quantitative PCR. RESULTS： The mutations in rdxA gene did not change in metronidazole resistant isolates treated with aspirin. The radioactivity of H pylori increased when treated with aspirin, indicating that aspirin improved the permeability of the outer membrane of H pylori. However, the expression of two OMP bands between 55 kDa and 72 kDa altered in the presence of aspirin.The expression of the mRNA of hopA, hopB, hopC, hopD, hopE and herA, hefB, hefC of H pylori did not change when treated with aspirin. CONCLUSION： Although aspirin increases the susceptibility of H pylori to metronidazole, it has no effect on the mutations of rdxA gene of Hpylori. Aspirin increases endocellular concentrations of antimicrobials probably by altering the OMP expression.

Simultaneous determination of metronidazole and tinidazole in plasma by using HPLC-DAD coupled with second-order calibration

A method using HPLC-DAD coupled with second-order calibration was developed to simultaneously determine metronidazole and tinidazole in plasma samples in this paper. The second-order calibration method based on APTLD （alternating penalty trilinear decomposition） algorithm was proposed to analyze the three-way HPLC-DAD data from both standard and prediction samples, which makes it possible that calibration can be performed even in the presence of unknown interferences with a simple and green chromatographic condition and short analysis time. The results showed that good recoveries were obtained although the chromatographic and spectral profiles of the analytes of interest as well as background were partially overlapped with each other in plasma samples.

Ultrasensitive electrochemical determination of metronidazole based on polydopamine/carboxylic multi-walled carbon nanotubes nanocomposites modified GCE

An ultrasensitive electrochemical sensor based on polydopamine/carboxylic multi-walled carbon nanotubes(MWCNTs à COOH) nanocomposites modified glassy carbon electrode(GCE) was presented in this work, which has been developed for highly selective and highly sensitive determination of an antimicrobial drug, metronidazole. The preparation of polydopamine/MWCNTs–COOH nanocomposites/GCE sensor is simple and possesses high reproducible, where polydopamine can be coated on the surface of MWCNTs–COOH via a simple electropolymerization process. Under optimized conditions, the proposed sensor showed ultrasensitive determination for metronidazole with a wide linear detection range from5 to 5000 mmol/dm^3 and a low detection limit of 0.25 mmol/dm^3(S/N=3). Moreover, the proposed sensor has been successfully applied for the quantitative determination of metronidazole in real drug samples. This work may provide a novel and effective analytical platform for determination of metronidazole in application of real pharmaceutical and biological samples analysis.

Screening for metronidazole-resistance associated gene fragments of H pylori by suppression subtractive hybridization

AIM： To screen for metronidazole （MTZ）-resistance associated gene fragments of H pylori by suppression subtractive hybridization （SSH）. METHODS： Five MTZ-resistant （tester, T） and 1 MTZ- susceptible （driver, D） clinical H pylori isolates were selected. Genomic DNAs were prepared and submitted to Rsa I digestion. Then two different adaptors were ligated respectively to the 5＇-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNAs. The specific inserts of tester strains were screened and MTZ-resistance related gene fragments were identified by dot blotting. RESULTS： Among the randomly selected 120 subtractive colonies, 37 DNA fragments had a different number of DNA copies （≥ 2 times） in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies （≥ 3 times）. Among the sequences obtained from the 17 DNA fragments, new sequences were found in 10 DNA fragments and duplicated sequences in 7 DNA fragments, representing respectively the sequences of depeptide ABC transporter periplasmic dipeptide-binding protein （dppA）, permease protein （dppB）, ribosomal protein S4 （rps4）, ribonuclease Ⅲ （rnc）, protease （pqqE）, diaminopimelate epimerase （dapF）, acetatekinase （ackA）, H pylori plasmid pHP51 and Hpylori gene 1334. CONCLUSION： Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant Hpylori.

A sensitive electrochemical detection of metronidazole in synthetic serum and urine samples using low-cost screen-printed electrodes modified with reduced graphene oxide and C60

Monitoring the concentration of antibiotics in body fluids is essential to optimizing the therapy and minimizing the risk of bacteria resistance,which can be made with electrochemical sensors tailored with appropriate materials.In this paper,we report on sensors made with screen-printed electrodes(SPE)coated with fullerene(C60),reduced graphene oxide(rGO)and Nafion(NF)(C60-rGO-NF/SPE)to determine the antibiotic metronidazole(MTZ).Under optimized conditions,the C60-rGO-NF/SPE sensor exhibited a linear response in square wave voltammetry for MTZ concentrations from 2.5×10^(-7) to 34×10^(-6) mol/L,with a detection limit of 2.1×10^(-7) mol/L.This sensor was also capable of detecting MTZ in serum and urine,with recovery between 94%and 100%,which are similar to those of the standard chromatographic method(HPLC-UV).Because the C60-rGO-NF/SPE sensor is amenable to mass production and allows for MTZ determination with simple principles of detection,it fulfills the requirements of therapeutic drug monitoring programs.

Ethnicity association of Helicobacter pylori virulence genotype and metronidazole susceptibility

AIM:To characterise the cag pathogenicity island in Helicobacter pylori(H.pylori) isolates by analysing the strains' vacA alleles and metronidazole susceptibilities in light of patient ethnicity and clinical outcome.METHODS:Ninety-five H.pylori clinical isolates obtained from patients with dyspepsia living in Malaysia were analysed in this study.Six genes in the cagPAI region(cagE,cagM,cagT,cag13,cag10 and cag67) andvacA alleles of theH.pylori isolates were identified by polymerase chain reaction.The isolates' metronidazole susceptibility was also determined using the E-test method,and the resistant gene was characterised by sequencing.RESULTS:More than 90% of the tested isolates had at least one gene in the cagPAI region,and cag67 was predominantly detected in the strains isolated from the Chinese patients,compared with the Malay and Indian patients(P < 0.0001).The majority of the isolates(88%) exhibited partial deletion(rearrangement) in the cagPAI region,with nineteen different patterns observed.Strains with intact or deleted cagPAI regions were detected in 3.2% and 8.4% of isolates,respectively.The prevalence of vacA s1m1 was significantly higher in the Malay and Indian isolates,whereas the isolates from the Chinese patients were predominantly genotyped as vacA s1m2(P = 0.018).Additionally,the isolates from the Chinese patients were more sensitive to metronidazole than the isolates from the Malay and Indian patients(P = 0.047).Although we attempted to relate the cagPAI genotypes,vacA alleles and metronidazole susceptibilities to disease outcome,no association was observed.The vacA alleles were distributed evenly among the strains with intact,partially deleted or deleted cagPAI regions.Interestingly,the strains exhibiting an intact cagPAI region were sensitive to metronidazole,whereas the strains with a deleted cagPAI were more resistant.CONCLUSION:Successful colonisation by different H.pylori genotypes is dependent on the host's genetic makeup and may play an important role in the clinical outcome.

Albendazole versus metronidazole treatment of adult giardiasis:An open randomized clinical study

AIM:To investigate the efficacy and tolerability of albendazole and metranidazole treatment in giardiasis. METHODS:The open comparative randomized trial was carried out prospectively from December 1999 to July 2001 in Duzce City of Turkey.The diagnosis was based on the presence of signs and symptoms compatible with giardiasis including a positive stool examination of giardia cysts or trophozoite.Metranidazole group consisted of 29 patients and was given metranidazole 500 mg,three times a day for 5 d and albendazole group was consisted of 28 patients and was given albendazole 400 mg/d for 5 d. RESULTS:There were no significant differences in demographical and therapeutical effects and patient's compliance between both groups.But side effects were seen more in metranidazole group than in albendazole group. CONCLUSION:Albendazole is as effective as metranidazole in adults' giardiasis.Albendazole has less side effect potentials than metranidazole in the treatment of giardiasis.

Determination of metronidazole in a rat stomach by HPLC for obtaining basic data of the eradication therapy of Helicobacter pylori

In the eradication therapy of Helicobacter pylori changes of antibiotics as these concentrations or amount in the stomach after oral administration were not clear. A simple and accurate method for determining the concentration of metronidazole （MTZ） in homogenate of rat stomach was developed in order to obtain basic data to design a pharmaceutical preparation having targeting ability to the surface of gastric-mucosa. This method included a deproteinization process by methanol, separation with reversed-phase high-performance liquid chromatography, and detection with an ultraviolet wavelength of 370 nm. Regression analysis showed that the method was linear over a standard curve nmge from 5 ug/mL to 2000 g/mL. The inter-day precision and accuracy values between thc ranges were 5.0% or better and -7.5 to 5.2%, respectively. The newly developed method was applied to an analysis of gastric samples after oral administration of MTZ at a dose of 5 mg/kg. It was found that the residual MTZ in the stomach was determined within 5 h after dosing. This method is useful for monitoring MTZ in stomach after its oral administration to rats.

An early report: a modified porphyrin-linked metronidazole targeting intracellular Porphyromonas gingivalis in cultured oral epithelial cells

Porphyromonas gingivalis （P. gingivalis） has a strong association with the pathogenesis of periodontal disease. Recurrence of periodontal disease following therapy is attributed to numerous factors, and of growing interest is the potential problem of intracellular bacteria that are able to persist and multiply within the host cell, thereby facilitating relapse of infection. The effect of antibiotic therapy in controlling P. gingivalis is questionable. Accordingly, while metronidazole is very effective against anaerobic extracellular P. gingivalis by disrupting the DNA of anaerobic microbial cells, this antibiotic does not effectively penetrate into mammalian cells to inhibit intracellular bacteria. Therefore in the present study, a modified porphyrin-linked metronidazole adducts, developed in our laboratory, was used to kill intracellular P. gingivalis. A series of experiments were performed, including cytotoxicity assays and cellular uptake of adducts by flow cytometry coupled with live cell imaging analysis, P. gingivalis invasion and elimination assays, and the analysis of colocalization of P. gingivalis and porphyrin-linked metronidazole by confocal laser scanning microscopy. Findings indicated that P. gingivalis and porphyrin-linked metronidazole were colocalized in the cytoplasm, and this compound was able to kill P. gingivalis intracellular with a sufficient culture time. This is a novel antimicrobial approach in the elimination of P. gingivalis from the oral cavity.

An Evaluation of Metronidazole Degradation in a Plasma-Assisted Rotating Disk Reactor Coupled with TiO2 in Aqueous Solution

Pollution involving pharmaceutical components in bodies of water is an increasingly serious environmental issue.Plasma discharge for the degradation of antibiotics is an emerging technology that may be relevant toward addressing this issue.In this work,a plasma-assisted rotating disk reactor(plasma-RDR)and a photocatalyst—namely,titanium dioxide(TiO_(2))—were coupled for the treatment of metronidazole(MNZ).Discharge uniformity was improved by the use of a rotating electrode in the plasma-RDR,which contributed to the utilization of ultraviolet(UV)light radiation in the presence of TiO_(2).The experimental results showed that the degradation efficiency of MNZ and the concentration of generated hydroxyl radicals respectively increased by 41%and 2.954 mg∙L^(-1) as the rotational speed increased from 0 to 500 r∙min^(-1).The synergistic effect of plasma-RDR plus TiO_(2) on the generation of hydroxyl radicals was evaluated.Major intermediate products were identified using three-dimensional(3D)excitation emission fluorescence matrices(EEFMs)and liquid chromatography-mass spectrometry(LC-MS),and a possible degradation pathway is proposed herein.This plasma-catalytic process has bright prospects in the field of antibiotics degradation.

Efficacy and Tolerability of Second-Line Metronidazole Triple Therapy Using Vonoprazan for Helicobacter pylori Eradication in Japan—Comparative Study: Vonoprazan vs. Proton Pump Inhibitors

Aim: To investigate the efficacy and tolerability of second-line metronidazole triple therapy with vonoprazan (VPZ) for Helicobacter pylori (H. pylori). Methods: We retrospectively reviewed medical records of patients who experienced clarithromycin triple therapy failure and were treated with second-line (20 mg VPZ (n = 274)/30 mg lansoprazole (n = 323) or 10 mg rabeprazole (n = 141) twice daily, 750 mg amoxicillin twice daily, 250 mg metronidazole twice daily for 7 days) eradication therapies. Successful eradication rates were compared between two groups: those receiving VPZ and those receiving a proton pump inhibitor (PPI). Adverse events were also investigated. Results: Successful second-line eradication rates according to ITT analysis and PP analysis, respectively, were 79.9% and 92.4% for VPZ therapy and 83.6% and 93.3% for PPI therapy. There were no significant differences between treatment groups. The eradication rates in those who had received first-line VPZ therapy previously according to ITT and PP analysis were 75.2% and 88.1%, respectively;in contrast, values were 82.5% and 95.4%, respectively, for those who had received first-line PPI therapy previously. In second-line therapy, the overall adverse event rate for VPZ therapy was the same as for PPI therapy. Conclusions: The efficacy and tolerability of metronidazole-containing second-line triple therapy with VPZ or a PPI were equivalent.

Introducing nitazoxanide as a promising alternative treatment for symptomatic to metronidazole-resistant giardiasis in clinical isolates

Objective:To identify the frequencies(F) of ferredoxin and nitroreductase mutations were identified on Iranian clinical isolates of Giardia lamblia in order to predict whether the nitazoxanide can be prescribed as suitable drug for symptomatic to metronidazoleresistant giardiasis.Methods:Forty Giardia lamblia isolates as of 38 symptomatic and two metronidazole-resistant patients were collected from Iran.DNAs were extracted and amplified by targeting ferredoxin and Gl NR genes.The amplicons were directly sequenced to determine gene mutations.Results:The various amino acid substitutions(F:20%,Haplotype diversity:0.891,Tajima's D:-0.44013) were identified by analyzing ferredoxin gene in four symptomatic and two resistant isolates.Only,two haplotypes(F:5%,HD:0.345; Tajima's D:0.77815) characterized in metronidazole-resistant isolates of Gl NR,however,no point mutations was found in symptomatic isolates.Conclusions:Non-synonymous mutations of ferredoxin oxidoreductase gene reduce translational regulatory protein's binding affinity which concludes reduction of ferredoxin expression and its activity.This leads to decrease in metronidazole drug delivery into the cells.Mutations in these isolates may lead to their resistance to metronidazole.No to low synonymous mutations of Gl NR demonstrates that nitazoxanide can be prescribed as promising alternative treatment for symptomatic to metronidazole-resistant giardiasis in Iranian clinical isolates.

Effect of Hypericum perforatum L. compared with metronidazole in bacterial vaginosis:a double-blind randomized trial

Objective:To compare effect of Hypericum perforatum(H.perforatum) vaginal gel with metronidazole on bacterial vaginosis(BV) in terms of initial response to treatment and preventing recurrence(primary outcomes) and also patient complaints(secondary outcomes).Methods:In this double-blind,double dummy trial,married women aged 18-49 with BV were randomized into two groups and administered 5 g of 3%H.perforatum and placebo of metronidazole(n=82),or 5 g of 0.75%metronidazole and placebo of H.peiforatum(n=80)vaginally for 5 d.Amsel criteria were used for diagnosis and assessing cure and recurrence of BV.The comparisons was done using Chi-square,Fisher's exact and logistic regression.Results:At 10-12 d.cure rate was 82%in the H.perforatum and 85%in metronidazole group(risk ratio 0.9.95%confidence interval 0.6 to 1.3).Among the cured women,recurrence rate was 9%in the H.perforatum and 13%in the metronidazole group at the 30-35 d visit(risk ratio 0.8,95%confidence interval 0.4 to 1.3),There was no statistically significant difference between the groups regarding any patient complaints,except itching which was less in H.perforatum group(5%vs.16%,P=0.018 at the first and 13%vs.43%,P<0.001 at the second follow-up).No significant adverse event was reported at any groups.Conclusions:H.peiforatum could be a good option for treatment of BV.However,further studies arc needed for its public use.

Synthesis and Crystal Structure of Metronidazole-derived Compound

A MET-OH derivative, MET-OTs 1, was designed, prepared and structurally characterized by single-crystal X-ray diffraction. X-ray structure analysis reveals that 1 crystallizes in the monoelinic system, space group P21/c, with a = 16.1178（14）, b = 7.5473（6）, c = 13.4161（11）A, V= 1520.3（2）A3,β=111.3210（10）°, Z= 4, Dc =1.421 g/cm^3 and F（000） = 680.

Influence of Some Physicochemical Exposure Factors on the Metronidazole Content of a Pharmaceutical Product: Flagyl®250 mg Tablet

The present work aims to study the influence of some physicochemical parameters (light, temperature, ethanol, bile salts, potassium hydroxide, hydrogen peroxide) on the content of Metronidazole (MTZ) contained in the reference substance (SR) and in a pharmaceutical specialty Flagyl® 250 mg tablet (FLG). The method developed was linear and accurate in accordance with USP 38 and the MTZ contents were obtained by UV-visible spectrophotometry at 278 nm. These contents ranged from 225 mg to 275 mg and were thus consistent with the concentration present in the proprietary medicines (250 mg). The study of the influence of physicochemical parameters on the MTZ content in SR and FLG showed that MTZ contents are unstable in the presence of Ethanol at 96˚, KOH at 0.1N and bile salts but also when the temperature is higher than 25˚C. However, they remain stable in the presence of light and H2O2 and undergo degradation in an acidic environment.

STUDY ON THE DETERMINATION OF METRONIDAZOLE IN HUMAN SERUM BY ADSORPTIVE STRIPPING VOLTAMMETRY

In Britton-Robinson buffer,metronidazole is preconcentreted on HHDE at 0.0 V(Vs.Ag- AgCI),An adsorptive stripping peak is observed at-0.62 V.Tho response is linear from 1x10^(-8)to 1×10^(-6)mol/L with 1.5 min accumulation.The method has been successfully applied to the deternation of metronidazole in human serum and formulations.

Complexes of cupric ion and tartaric acid enhanced calcium peroxide Fenton-like reaction for metronidazole degradation

To surmount the obstacles of traditional Fenton method and synchronously utilize Cu^(2+)and polyphenol in water,an improved Fenton-like reaction applying calcium peroxide(CaO_(2))as H_(2)O_(2)source and regulating by the complex of Cu^(2+)-tartaric acid(TA,a representative of polyphenol)was constructed.A typical antibiotic,metronidazole(MTZ)could be effectively eliminated by the Cu^(2+)/TA/CaO_(2)system,and the optimized parameters were as follows:0.1 mmol/L Cu^(2+),2 mmol/L TA,2 mmol/L CaO_(2),and initial pH5.UV spectrum confirmed the formation of Cu^(2+)-TA complex,which promoted the Cu^(2+)/Cu+circulation through decreasing the Cu^(2+)/Cu^(+) couple redox potential,which further enhanced the H_(2)O_(2)decomposition and the formation of reactive species.Hydroxyl radical was dominant for MTZ degradation,followed by oxygen and superoxide radical.The degradation intermediates of MTZ were detected and their evolution way was speculated.Furthermore,the ternary process showed a wide p H tolerance(3–8)for removing MTZ and broad applicability for eliminating other dyes and antibiotics.This work provided a reference for Cu-based Fenton-like strategy for organic wastewater settlement.

A New Approach for the Determination of Metronidazole Using Switchable Solvent Coupled with Au-MWCNTs-Modified Electrochemical Sensor

In this work,a new liquid-phase microextraction method coupled with an electrochemical technique using a switchable solvent was proposed for the determination of metronidazole.The extraction solvent used was dipropylamine(DPA),which exhibited switchable hydrophilicity.This means that it can become miscible or immiscible when in contact with carboxylic acids in the aqueous sample.The metronidazole was then measured using a glassy carbon electrode that was modified with Au-multiwalled carbon nanotubes(MWCNTs).The eff ect of diff erent parameters such as the type and amount of trigger,the addition of salt,and the volume of extraction solvent on the e fficiency of switchable hydrophilic solvent-based liquid-phase microextraction(SHS-LPME)was investigated using the one-factor-at-a-time method.After optimizing the conditions,the linear calibration curve was obtained in the concentration range of 0.005–250μmol/L.Accordingly,the limit of quantification(LOQ)of 0.005μmol/L and the limit of detection(LOD)of 0.0015μmol/L were obtained,respectively.