Metronidazole Susceptibility Research Articles

Antimicrobial Susceptibility and Resistance Patterns among Helicobacter pylori Strains from The Gambia, West Africa

Helicobacter pylori is a globally important and genetically diverse gastric pathogen that infects most people in developing countries. Eradication efforts are complicated by antibiotic resistance, which varies in frequency geographically. There are very few data on resistance in African strains. Sixty-four Gambian H. pylori strains were tested for antibiotic susceptibility. The role of rdxA in metronidazole (Mtz) susceptibility was tested by DNA transformation and sequencing; RdxA protein variants were interpreted in terms of RdxA structure. Forty-four strains (69%) were resistant to at least 8 μg of Mtz/ml. All six strains from infants, but only 24% of strains from adults, were sensitive (P = 0.0031). Representative Mtz-resistant (Mtz(r)) strains were rendered Mtz susceptible (Mtz(s)) by transformation with a functional rdxA gene; conversely, Mtz(s) strains were rendered Mtz(r) by rdxA inactivation. Many mutations were found by Gambian H. pylori rdxA sequencing; mutations that probably inactivated rdxA in Mtz(r) strains were identified and explained using RdxA protein's structure. All of the strains were sensitive to clarithromycin and erythromycin. Amoxicillin and tetracycline resistance was rare. Sequence analysis indicated that most tetracycline resistance, when found, was not due to 16S rRNA gene mutations. These data suggest caution in the use of Mtz-based therapies in The Gambia. The increasing use of macrolides against respiratory infections in The Gambia calls for continued antibiotic susceptibility monitoring. The rich variety of rdxA mutations that we found will be useful in further structure-function studies of RdxA, the enzyme responsible for Mtz susceptibility in this important pathogen.

Resistance rates of metronidazole and other antibacterials in Helicobacter pylori from previously untreated patients in Norway

The aim of the study was to describe the antimicrobial resistance rate of Helicobacter pylori isolated from previously untreated patients in Norway, including the application of two different methods for the determination of metronidazole susceptibility. Altogether 102 isolates obtained in 2008 and 2009 from previously untreated patients suspected of H. pylori related disease, were examined applying a standardized European study protocol. The activity of amoxicillin, tetracycline, clarithromycin, metronidazole, rifabutin and levofloxacin was recorded after an incubation period of 72-96 h in a microaerobic atmosphere. Strains resistant to metronidazole were re-examined for metronidazole resistance applying anaerobic conditions for the first 24 h. None of the isolates were resistant to amoxicillin or tetracycline, whereas 5, 9% were resistant to clarithromycin and 22, 5% resistant to metronidazole tested conventionally. Applying local standards the metronidazole resistance rate fell to 7, 8%, highlighting the importance of the methodology applied for metronidazole susceptibility testing.

Mycoplasma hominis in Cuban Trichomonas vaginalis isolates: Association with parasite genetic polymorphism

Trichomonas vaginalis can be naturally infected with intracellular Mycoplasma hominis. This bacterial infection may have implications for trichomonal virulence and disease pathogenesis. The objective of the study was to report the presence of M. hominis in Cuban T. vaginalis isolates and to describe the association between the phenotype M. hominis infected with RAPD genetic polymorphism of T. vaginalis. The Random Amplified Polymorphic DNA (RAPD) technique was used to determine genetic differences among 40 isolates of T. vaginalis using a panel of 30 random primers and these genetic data were correlated with the infection of isolates with M. hominis. The trees drawn based on RAPD data showed no relations with metronidazole susceptibility and significantly association with the presence of M. hominis (P=0.043), which demonstrates the existence of concordance between the genetic relatedness and the presence of M. hominis in T. vaginalis isolates. This result could point to a predisposition of T. vaginalis for the bacterial enters and/or survival.

Antibiotic resistance and adhesion potential of Bacteroides fragilis clinical isolates from Cape Town, South Africa

The minimum inhibitory concentrations of 23 Bacteroides fragilis clinical isolates from Cape Town, South Africa, were established using the E-test method. Eight percent of the strains were found to be highly resistant to metronidazole (≥256 mg/L) imipenem and cefoxitin. This is an 8% increase in resistance compared to the previous metronidazole susceptibility screening performed in South Africa in 1998. Clindamycin was the most effective antibiotic with all strains showing sensitivity. Most of the strains (65%) were tetracycline resistant, while one strain, B. fragilis GSH15, showed multidrug resistance to metronidazole, imipenem, cefoxitin and tetracycline. PCR screening revealed that none of the strains contained any of the published nim genes. The particle agglutination assay was employed to determine the ability of the isolates to bind the ECM components fibronectin, laminin, mucin and collagen. This revealed that 78% of the clinical isolates adhered to all four ECM components to varying extents, with the strongest being to laminin and weakest to mucin and collagen Type I.

Comparative drug susceptibility study of five clonal strains of Trichomonas vaginalis in vitro

Objective To produce comparative data on a group of Trichomonas vaginalis clonal strains with varied drug responses using identical methods and materials. Methods Five clonal strains of Trichomonas vaginalis were isolated from reference strain using agar plate technique. The variability of growth kinetic and susceptibility of clonal strain to metronidazole, tinidazole, satranidazole and nitazoxanide were observed in 96 well microtitre plate. Results Among these clonal strains there was a good correlation between rates of growth with the relative susceptibility of the strains to drugs in vitro. Regarding metronidazole, tinidazole and satranidazole susceptibility, different degrees of susceptibility were determined. However, no difference in nitazoxanide susceptibility was found between the clonal strain tested and a reference strain. Conclusions This is the first description of biological variability in clonal stock of Trichomonas vaginalis. Different degrees of drug susceptibility were determined among clonal strains tested. Further studies will be necessary to ascertain the importance of this variability in clinical infection.

Genetic Relatedness of Trichomonas vaginalis Reference and Clinical Isolates

We have determined the metronidazole susceptibility status of 20 Trichomonas vaginalis isolates from American Type Culture Collection (ATCC) and assessed the level of genetic relatedness in these isolates using 32 additional T. vaginalis clinical isolates for comparison. These ATCC isolates are commonly used as reference strains in T. vaginalis research and this information provides a rational basis for selection of reference strains for use in comparative studies of T. vaginalis phenotypic and clinical characteristics.

Recent trends in primary antimicrobial resistance of Helicobacter pylori in Finland

The antimicrobial susceptibility of Helicobacter pylori is an important predictor of the success of eradication therapy. To evaluate recent changes in primary antimicrobial resistance of H. pylori isolated from Finnish patients, the clinical records of H. pylori-positive patients referred for endoscopy to Herttoniemi Hospital (Helsinki, Finland) during 2000–2008 were investigated retrospectively. Stored H. pylori strains from 505 patients without previous eradication therapy were tested for clarithromycin, metronidazole, levofloxacin, tetracycline and amoxicillin susceptibility by Etest. Data on local consumption of antimicrobials were collected and correlations between consumption and resistance were calculated. During the 9-year study period, metronidazole resistance was high (range 29–59%, overall 41%). After an initial increase in clarithromycin resistance (0% in 2000 to 16% in 2003), resistance to clarithromycin decreased to 4% in 2008. No significant correlation was detected between consumption of macrolides and resistance of clarithromycin. Resistance to levofloxacin varied between 0% and 12%. Primary metronidazole resistance in H. pylori is at a high level, however levofloxacin and clarithromycin resistances are still at a reasonable level. Thus, primary clarithromycin resistance in H. pylori in Finland has not become such a problem as in many other countries. Primary resistance to the antimicrobials studied varied considerably from year to year.

Mycoplasma hominis infection of Trichomonas vaginalis is not associated with metronidazole-resistant trichomoniasis in clinical isolates from the United States

Trichomonas vaginalis is a protozoan parasite that is the cause of the most common non-viral sexually transmitted disease, trichomoniasis. Metronidazole and tinidazole are the only drugs approved for treatment of T. vaginalis infections in the USA. However, drug resistance exists and some patients are allergic to these medications. Furthermore, the exact mechanism of metronidazole resistance remains undefined and current testing methods require several weeks before results are available. Identification of the mechanism of drug resistance may lead to the development of molecular tools to detect drug resistance, and quicker results for clinical treatment. In a recent study, Chinese T. vaginalis isolates that were polymerase chain reaction (PCR) positive for Mycoplasma hominis DNA demonstrated greater in vitro resistance to metronidazole than isolates with no evidence of M. hominis infection. To evaluate this finding in isolates from a distinct epidemiologic setting, we tested 55 T. vaginalis isolates collected from patients in the USA through the Centers for Disease Control and Prevention metronidazole susceptibility testing service. One half of the isolates demonstrated resistance to metronidazole by an in vitro sensitivity assay. Of the metronidazole-resistant T. vaginalis isolates, 18% were PCR positive for M. hominis, as were 22% of the metronidazole-susceptible T. vaginalis isolates (p = 0.746). We also observed no change in metronidazole sensitivity of two infected T. vaginalis isolates after they were cleared of their M. hominis infection by culturing the isolates in antibiotics. Thus, M. hominis infection of USA T. vaginalis isolates did not correlate with in vitro resistance to metronidazole.

Emergence of reduced susceptibility to metronidazole in Clostridium difficile

Antimicrobial treatment for Clostridium difficile infection (CDI) has typically been metronidazole, although reports have questioned the efficacy of this option. We screened recently isolated C. difficile (2005-06) for susceptibility to metronidazole and compared results for historic isolates (1995-2001). C. difficile ribotypes 001 (n = 86), 106 (n = 81) and 027 (n = 48) and isolates from the 10 other most prevalent ribotypes in Leeds (n = 57) were screened using spiral gradient endpoint analysis (SGE). C. difficile with metronidazole SGE MICs > or = 6 mg/L were analysed further by agar incorporation and Etest. Multiple-locus variable-number tandem-repeat analysis (MLVA) typing was performed for 28 C. difficile isolates. No reduced metronidazole susceptibility was observed in C. difficile ribotypes 106 and 027 (geometric mean SGE MICs 1.11 and 0.90 mg/L, respectively). In contrast, 21 (24.4%) C. difficile ribotype 001 demonstrated reduced susceptibility to metronidazole (geometric mean SGE MICs 3.51 mg/L, P < 0.001). Variations in susceptibility were observed relating to the method and media, but increased metronidazole MICs were confirmed by an agar incorporation method. Geometric mean agar incorporation MICs for historic C. difficile ribotype 001 (n = 72) were 1.03 (range 0.25-2) mg/L compared with 5.94 (4-8) mg/L (P < 0.001) for recent isolates displaying reduced metronidazole susceptibility. MLVA typing revealed two clonal complexes of C. difficile with reduced susceptibility to metronidazole. We have demonstrated the emergence of reduced susceptibility to metronidazole in 24.4% of the recent C. difficile ribotype 001 isolates from our institution. Our observations could have implications in the clinical setting due to the poor penetration of metronidazole into the colon.

Surveillance de la résistance aux antibiotiques des anaérobies stricts à Gram négatif

Material and method Using an agar reference method (Norma M11-A5, National Committee for Clinical and Laboratory Standards) the minimal inhibitory concentrations of nine antibiotics were determined for 376 anaerobic strains. The following strains were investigated: 254 Bacteroides fragilis group (including 143 B. fragilis), 122 other gram-negative anaerobes ( Bacteroides spp. , Prevotella, Fusobacterium, Porphyromonas, Suterella, Desulfomonas, Veillonella). Results In the B. fragilis group resistance rates were: coamoxyclav 2.8%, ticarcillin 27.5%, ticarcillin–clavulanic acid 1.9%, piperacillin–tazobactam 1.9%, cefoxitin 6.2%, imipenem 0.8%, clindamycin 28.3%, respectively. Based on previous studies, resistance to imipenem remained low in 2003 and was only observed for B. fragilis. Resistance to clindamycin was maintained around 25%. No metronidazole resistance was observed, but decreased susceptibility was found for B. fragilis, B. merdae and Prevotella, as in 4.3% of gram-negative anaerobes. Discussion This study confirms the high resistance rate of gram-negative anaerobes to clindamycin, the efficient activity of imipenem, β-lactam/β-lactamase inhibitor combinations and metronidazole. However, reduced metronidazole susceptibility seems to be increasing.

Activity of Pradofloxacin against Porphyromonas and Prevotella spp. Implicated in Periodontal Disease in Dogs: Susceptibility Test Data from a European Multicenter Study

Collaborating veterinarians from five European countries collected subgingival bacterial samples from dogs exhibiting clinical periodontal disease. Sterile endodontic paper points were used for collection of the samples, which were transported to a central laboratory for susceptibility testing. Anaerobic bacteria were isolated and Porphyromonas and Prevotella isolates identified to the species level; susceptibility to pradofloxacin and metronidazole was determined using the CLSI agar dilution methodology. A total of 630 isolates, 310 of Porphyromonas spp. and 320 of Prevotella spp., were isolated. Pradofloxacin MIC data for all isolates were in the range of < or =0.016 to 1 microg/ml, the overall MIC(50) was 0.062, and the overall MIC(90) was 0.25 microg/ml. There were no differences in activity against Porphyromonas and Prevotella isolates or in the pradofloxacin susceptibility distributions from the different European countries. All isolates were within the wild-type distribution and were fully susceptible to pradofloxacin. Metronidazole was also highly active against these strains: 316 of 320 Prevotella strains (98.8%) and 309 of 310 Porphyromonas strains (99.7%) were susceptible (MICs of < or =8 microg/ml). However, three Prevotella strains had intermediate metronidazole susceptibility (MICs of 16 microg/ml), while one Prevotella and one Porphyromonas strain were metronidazole resistant (MICs of 128 and 256 microg/ml, respectively). Pradofloxacin, a novel broad-spectrum fluoroquinolone, demonstrates a high degree of antianaerobic activity against strains isolated from clinical cases of periodontal disease and shows activity against metronidazole-resistant isolates. The broad-spectrum activity of pradofloxacin makes it a suitable candidate for the treatment of periodontal disease in dogs.

Early repeated infections with Trichomonas vaginalis among HIV-positive and HIV-negative women.

The purpose of the study was to examine whether early repeated infections due to Trichomonas vaginalis among human immunuodeficiency virus (HIV)-positive and HIV-negative women are reinfections, new infections, or cases of treatment failure. Women attending an HIV outpatient clinic and a family planning clinic in New Orleans, Louisiana, who had culture results positive for T. vaginalis were treated with 2 g of metronidazole under directly observed therapy. At 1 month, detailed sexual exposure and sexual partner treatment information was collected. Isolates from women who had clinical resistance (i.e., who tested positive for a third time after treatment at a higher dose) were tested for metronidazole susceptibility in vitro. Of 60 HIV-positive women with trichomoniasis, 11 (18.3%) were T. vaginalis positive 1 month after treatment. The 11 recurrences were classified as 3 probable reinfections (27%), 2 probable infections from a new sexual partner (18%), and 6 probable treatment failures (55%); 2 of the 6 patients who experienced probable treatment failure had isolates with mild resistance to metronidazole. Of 301 HIV-negative women, 24 (8.0%) were T. vaginalis positive 1 month after treatment. The 24 recurrences were classified as 2 probable reinfections (8%) and 22 probable treatment failures (92%); of the 22 patients who experienced probable treatment failure, 2 had strains with moderate resistance to metronidazole, and 1 had a strain with mild resistance to metronidazole. HIV-positive women were more likely to have sexual re-exposure than were HIV-negative women, although the rate of treatment failure was similar in both groups. High rates of treatment failure among both HIV-positive and HIV-negative women indicate that a 2-g dose of metronidazole may not be adequate for treatment of some women and that rescreening should be considered.

Comparison of efficacies of dual therapy and triple therapy using rabeprazole in second‐line eradication of Helicobacter pylori in Japan

SummaryBackgroundThe only authorized second‐line Helicobacter pylori regimen in Japan is proton pump inhibitor + amoxycillin + clarithromycin. However, it has been reported that this second‐line regimen is not effective. In this study, we evaluated the efficacy of dual and triple eradication therapies using rabeprazole as second‐line H. pylori eradication regimens.AimTo evaluate the efficacy of dual and triple eradication therapies using rabeprazole as second‐line H. pylori eradication therapy.MethodsSixty‐two H. pylori‐positive patients with first‐line eradication failure were randomly assigned to two groups. The RAM group was administered rabeprazole 20 mg + amoxycillin 1500 mg + metronidazole 500 mg daily for 1 week. The RA group was administered rabeprazole 40 mg + amoxycillin 2000 mg daily for 2 weeks. Eradication of H. pylori infection was determined by 13C‐urea breath testing at 8 weeks after completion of treatment. Prior to treatment, amoxycillin, clarithromycin and metronidazole susceptibility, and CYP2C19 phenotype status were determined.ResultsEradication rates for the RAM and RA groups were 97% and 74%, respectively. Eradication rates were not influenced by CYP2C19 phenotype in either group. Eradication rates for clarithromycin‐resistant patients were 100% in the RAM group and 77% in the RA group.ConclusionsOne week with RAM therapy and 2 weeks with RA therapy were effective as second‐line eradication therapy for H. pylori infection; moreover, RAM was more effective than RA therapy.

Comparison of efficacies of dual therapy and triple therapy using rabeprazole in second‐line eradication of Helicobacter pylori in Japan

SummaryBackgroundThe only authorized second‐line Helicobacter pylori regimen in Japan is proton pump inhibitor + amoxycillin + clarithromycin. However, it has been reported that this second‐line regimen is not effective. In this study, we evaluated the efficacy of dual and triple eradication therapies using rabeprazole as second‐line H. pylori eradication regimens.AimTo evaluate the efficacy of dual and triple eradication therapies using rabeprazole as second‐line H. pylori eradication therapy.MethodsSixty‐two H. pylori‐positive patients with first‐line eradication failure were randomly assigned to two groups. The RAM group was administered rabeprazole 20 mg + amoxycillin 1500 mg + metronidazole 500 mg daily for 1 week. The RA group was administered rabeprazole 40 mg + amoxycillin 2000 mg daily for 2 weeks. Eradication of H. pylori infection was determined by 13C‐urea breath testing at 8 weeks after completion of treatment. Prior to treatment, amoxycillin, clarithromycin and metronidazole susceptibility, and CYP2C19 phenotype status were determined.ResultsEradication rates for the RAM and RA groups were 97% and 74%, respectively. Eradication rates were not influenced by CYP2C19 phenotype in either group. Eradication rates for clarithromycin‐resistant patients were 100% in the RAM group and 77% in the RA group.ConclusionsOne week with RAM therapy and 2 weeks with RA therapy were effective as second‐line eradication therapy for H. pylori infection; moreover, RAM was more effective than RA therapy.

Use of Trichomonas vaginalis Clinical Isolates to Evaluate Correlation of Gene Expression and Metronidazole Resistance

We investigated whether variations in gene expression of enzymes associated with anaerobic resistance of laboratory-derived strains of Trichomonas vaginalis could be detected in a group of 28 clinical isolates with variations in metronidazole sensitivity. We compared isolates by real-time PCR because this method allows for highly sensitive quantification of mRNA and for evaluation of several genes simultaneously. We found that PFOR gene A mRNA levels were highly correlated with PFOR gene B levels, as well as the D subunit of malic enzyme and ferrodoxin. Ferrodoxin mRNA expression was also significantly correlated with that of malic enzyme and hydrogenase. However, when we evaluated relationships between these enzymes and resistance to metronidazole, we found no significant correlations between aerobic or anaerobic in vitro sensitivity to drug and mRNA levels of any of the enzymes tested. Similarly, using a Student's t-test, no significant differences in enzyme mRNA levels were observed between isolates separated by metronidazole resistance or susceptibility. The lack of correlation between gene expression and resistance or susceptibility could be the result of differences in expression at the protein level or because other biochemical pathways or genes are involved in the resistance observed in clinical settings.

DNA sequence analysis of rdxA and frxA from paired metronidazole-sensitive and -resistant Helicobacter pylori isolates obtained from patients with heteroresistance

Genomic variations of rdxA and frxA genes in eight pairs of metronidazole (MTZ)-sensitive and -resistant isolates of Helicobacter pylori were investigated. The paired strains from each single biopsy had identical lspA- glmM restriction fragment length polymorphism profiles, and the differences in MTZ susceptibility were mostly accounted for by mutations in the rdxA gene. Truncation of RdxA is associated with a minimum inhibitory concentration of 64–128 mg/L. Early truncation of FrxA was observed both in susceptible and resistant isolates of seven paired strains. Inactivation of frxA might play a significant role only in one low-level MTZ-resistant isolate where a missense mutation was found in the rdxA gene.

Accuracy of E-test for Metronidazole Susceptibility in H. Pylori

Accuracy of E-test for Metronidazole Susceptibility in H. Pylori

Nitroimidazole resistance genes (nimB) in anaerobic Gram-positive cocci (previously Peptostreptococcus spp.).

To investigate metronidazole resistance and the prevalence of nitroimidazole (nim) genes in clinically isolated anaerobic Gram-positive cocci. Metronidazole susceptibility was determined in 99 strains of anaerobic Gram-positive cocci and PCR amplification for the nim gene carried out on 61 strains (metronidazole MIC > or =0.5 mg/L). The nimB gene was detected in 34% (21/61) of the strains. These included two highly resistant Finegoldia magna strains (MICs >128 mg/L). The nimB gene was, however, also demonstrated in 90% (19/21) of susceptible strains. Although the nimB gene may be implicated in the high-level metronidazole resistance in 2 F. magna strains, the alarmingly high prevalence of the nimB gene in anaerobic Gram-positive cocci cannot be directly associated with resistance and the possibility of a silent nimB gene should be considered.

Targeted gene replacement of a ferredoxin gene in Trichomonas vaginalis does not lead to metronidazole resistance.

Ferredoxin, Fd, is often deficient in metronidazole-resistant strains of Trichomonas vaginalis and is thought to be necessary for drug activation. To directly test whether Fd is essential for metronidazole susceptibility, gene replacement technology has been developed for T. vaginalis. The selectable marker gene neomycin phosphotransferase (NEO) flanked by approximately 2.6 and approximately 2.0 kBp of the Fd 5' and 3' flanking regions (pKO-FD-NEO) was introduced into cells on linear DNA and selected for NEO gene expression. Stable transformants were shown to contain the NEO gene in the Fd locus and to have completely lost the Fd gene. Northern and immunoblot analyses confirm the loss of Fd mRNA and protein in pKO-FD-NEO cells. Analyses of the activity of hydrogenosomal proteins in Fd KO cells show a fourfold increase in hydrogenase activity and a 95% decrease in pyruvate/ferredoxin oxidoreductase (PFO) activity. In contrast, PFO and hydrogenase mRNA levels are unchanged. Surprisingly, Fd KO cells are not resistant to metronidazole under aerobic or anaerobic conditions. These cells are capable of producing molecular hydrogen, albeit at 50% the level of the parental strain, demonstrating that the Fd gene product eliminated in KO cells is neither necessary for hydrogen production nor metronidazole activation. Together these data indicate the presence of unidentified Fds or flavodoxins capable of drug activation or an unidentified mechanism that does not require either PFO or Fd for metronidazole activation.

Is epsilometer test (E-t) to determine metronidazole susceptibility (MS) of H. pylori (Hp) really unreliable?

Is epsilometer test (E-t) to determine metronidazole susceptibility (MS) of H. pylori (Hp) really unreliable?