Metronidazole Susceptibility Research Articles

Molecular Study: Nitroreductase Enzyme and Nitroimmidazole Resistance Genes (Nim Genes) Effect on AMR to Metronidazole in Clostridium difficile

Nitroreductase enzymes and Nim genes have been observed to be a factor predisposing to reduced susceptibility to metronidazole in anaerobic bacteria. Therefore, this study was determined the effect of nitroreductase enzymes and nim genes in metronidazole susceptibility in UK C. difficile isolates. Three UK C. difficile ribotypes 027, 001, 106 including metronidazole reduced susceptible (CDRM) and metronidazole susceptible (CDSM) C. difficile strains, and 2 control strains from ribotype 010 (CDRM) and 038 (CDSM) were used. Control strain C. perfringens ATCC 3626 previously identified to be nitroreductase positive, and positive control for nimB which was Bacteroides fragilis strain BF8 was also included in this research. These selected strains were observed for their nitroreductase activity and presence of Nim genes. C. perfringens ATCC 3626 demonstrated nitroreductase activity but was absent in C. difficile control. Three CDRM strains (001 and 106 ribotype) demonstrated nitoreductase activity. None of the CDSM strains analysed showed detectable nitroreductase activity. The nim gene PCR amplification was only observed in the nim positive control strain BF8 (about 400 bp). All samples tested including ribotype 001 027 and 106 CDSM and CDRM strains showed no nimPCR product. In conclusion, despite the nitroreductase result obtained, inconsistencies observed during the assay showed methodology issues, thus using radio labelled metronidazole and monitoring the decline in the radiolabel signal proportional to the level of nitroreduction, maybe a preferred method in correlating nitroreductase activity to the CDRM phenotype. Also, this research observed the absence of nim genes in C. difficile UK ribotypes irrespective of metronidazole susceptibility patterns, depicting the inability of the universal primers for nimA-NimE to amplify a potential nim gene in C. difficile UK ribotypes. The universal primers were previously reported incapable of amplifying nimJ due to a different nucleotide sequence coding for same proteins as the universal primers.

MALDI - TOF MS for the identification of obligate anaerobes and metronidazole susceptibility of anaerobic Gram negative bacilli

MALDI-TOF MS, though has facilitated rapid and accurate anaerobe identification, the problem of rising metronidazole resistance amongst the members of Bacteroides fragilis group is cause for concern. In this one-year study period,152 anaerobic Gram negative bacilli and 60 Gram positive anaerobes were isolated from 167 samples obtained from clinically suspected anaerobic infections. Bacteroides fragilis accounted for 56 % of the total anaerobic GNB and Peptoniphilus asacchrarolyticus was the most commonly isolated Gram positive cocci. All isolates were identified by the MALDI–TOF MS except one isolate each of Clostridium and Peptostreptococcus. E-test for members of Bacteroides fragilis group, demonstrated 26.8 % metronidazole resistance.

Rapid Antimicrobial Susceptibility Test of Helicobacter pylori to Metronidazole via Single-Cell Raman Spectrometry.

Metronidazole is a first-line antibiotic to treat Helicobacter pylori infections. However, the Clinical Laboratory Standards Institute guidelines recommend against using antimicrobial susceptibility test (AST) to test metronidazole resistance, due to the unreliable predictive power which can result in treatment failure. The aim of this study was to establish an 8-h, metabolic-phenotype based AST for H. pylori metronidazole susceptibility using D2O-probed Raman microspectroscopy. Minimal inhibitory concentration (MIC) measured by conventional AST (E-test) were compared with expedited MIC via metabolic activity (eMIC-MA) for 10 H. pylori isolates. Raman barcodes of cellular-response to stress (RBCS) incorporating protein and carbohydrate Raman bands, were utilized to identify a biomarker to distinguish metronidazole susceptibility. Specifically, eMIC-MA produces metronidazole susceptibility results showing 100% agreement with E-test, and determines the bactericidal dosage for both high- and low-level resistant H. pylori strains. In addition, RBCS not just reliably distinguish between metronidazole-susceptible and -resistant strains, but reveal their distinct mechanisms in bacterial responses to metronidazole. The speed, accuracy, low cost, and rich information content that reveals the mode-of-action of drugs suggest the method's value in guiding metronidazole prescriptions for H. pylori eradication and in rapid screening based on drug-resistance mechanism.

New functions of pirin proteins and a 2-ketoglutarate: Ferredoxin oxidoreductase ortholog in Bacteroides fragilis metabolism and their impact on antimicrobial susceptibility to metronidazole and amixicile.

The understanding of how central metabolism and fermentation pathways regulate antimicrobial susceptibility in the anaerobic pathogen Bacteroides fragilis is still incomplete. Our study reveals that B. fragilis encodes two iron-dependent, redox-sensitive regulatory pirin protein genes, pir1 and pir2. The mRNA expression of these genes increases when exposed to oxygen and during growth in iron-limiting conditions. These proteins, Pir1 and Pir2, influence the production of short-chain fatty acids and modify the susceptibility to metronidazole and amixicile, a new inhibitor of pyruvate: ferredoxin oxidoreductase in anaerobes. We have demonstrated that Pir1 and Pir2 interact directly with this oxidoreductase, as confirmed by two-hybrid system assays. Furthermore, structural analysis using AlphaFold2 predicts that Pir1 and Pir2 interact stably with several central metabolism enzymes, including the 2-ketoglutarate:ferredoxin oxidoreductases Kor1AB and Kor2CDAEBG. We used a series of metabolic mutants and electron transport chain inhibitors to demonstrate the extensive impact of bacterial metabolism on metronidazole and amixicile susceptibility. We also show that amixicile is an effective antimicrobial against B. fragilis in an experimental model of intra-abdominal infection. Our investigation led to the discovery that the kor2AEBG genes are essential for growth and have dual functions, including the formation of 2-ketoglutarate via the reverse TCA cycle. However, the metabolic activity that bypasses the function of Kor2AEBG following the addition of phospholipids or fatty acids remains undefined. Overall, our study provides new insights into the central metabolism of B. fragilis and its regulation by pirin proteins, which could be exploited for the development of new narrow-spectrum antimicrobials in the future.

Antimicrobial Resistance Molecular Mechanisms of Helicobacter pylori in Jordanian Children: A Cross-Sectional Observational Study

H. pylori antimicrobial resistance causes increasing treatment failure rates among H. pylori gastritis in children. This study investigates the molecular mechanisms of H. pylori antimicrobial resistance among Jordanian children. Demographic, clinical, and laboratory data were recorded for children referred to Prince Hamzah Hospital. Clarithromycin, Metronidazole, and Levofloxacin susceptibility were tested via E-test. Clarithromycin-related mutations were investigated using Real-Time (RT)-PCR and Levofloxacin resistance was analyzed with DNA sequencing of the gyrA gene. 116 children were recruited, including 55.2% females and 55.2% in the age range of 10.1 to 14 years. A total of 82.7% were naïve to eradication therapy. H. pylori positivity was 93.9%, 89.6%, 61.7%, and 84.3% according to Rapid Urease Test, histology, culture, and RT-PCR, respectively. Resistance rates were 25.9% for Clarithromycin, 50% for Metronidazole, and 6.9% for Levofloxacin via E-test. A2142G or A2143G or a combination of both mutations concerning Clarithromycin resistance were documented in 26.1% of samples, while mutations in gyrA gen-related to Levofloxacin resistance were reported in 5.3% of samples. Antibiotic resistance was significantly affected by abdominal pain, anemia, hematemesis, and histological findings (p < 0.05). H. pylori resistance was documented for Metronidazole and Clarithromycin. RT-PCR for H. pylori identification and microbial resistance determination are valuable alternatives for cultures in determining antimicrobial susceptibility.

Prevalence and metronidazole resistance of Trichomonas vaginalis among Japanese women in 2021

ObjectivesTrichomonas vaginalis is the most prevalent sexually transmitted parasite worldwide. However, no surveillance system exists to monitor T. vaginalis cases and drug resistance in Japan. MethodsCervical cytology vaginal swabs were collected from women with and without suspected symptoms of T. vaginalis infection; these swabs were used for the detection of T. vaginalis, human papillomavirus (HPV), and Candida albicans using specific polymerase chain reaction. Clinical isolates of T. vaginalis were subjected to metronidazole susceptibility tests using the previously reported minimal lethal concentration (MLC) and newly established half-maximal inhibitory concentration (IC50) values. ResultsThe prevalence of T. vaginalis in the study population was 4.2% (5/119; 95% confidence interval [Cl], 1.5–9.7). Additionally, asymptomatic infection constituted 60% (3/5) of all cases of T. vaginalis infection. All T. vaginalis-positive patients were coinfected with HPV but not C. albicans. Five clinical T. vaginalis isolates showed metronidazole susceptibility, which was evaluated using MLC values. The quantitative IC50 values revealed that two of these clinical isolates exhibited a decreased metronidazole susceptibility. ConclusionThis is the first study to demonstrate the prevalence of T. vaginalis in Japanese women. The IC50 values of metronidazole against T. vaginalis enabled the precise and quantitative evaluation of metronidazole-susceptible T. vaginalis.

Molecular study on metronidazole resistance in Bacteroides fragilis group isolates from a South Indian tertiary care center

ObjectiveBacteroides species are an important part of human intestinal microbiota. They can cause infections of significant mortality and morbidity when moved out of their niche in the gut. The cornerstone drug for prophylaxis and therapy, metronidazole, is exhibiting signs of resistance, which are frequently attributed to nitroimidazole (nim) resistance genes. The aim of this study was to use Epsilometer test (E-test) to assess the metronidazole susceptibility and conventional PCR methodology to map the distribution of nim genes in Bacteroides fragilis group (BFG) isolates. MethodsMALDI-TOF MS was used to identify BFG isolates. Using the E-test methodology, metronidazole minimum inhibitory concentrations (MICs) were determined. The presence of nim genes in these isolates were checked by conventional PCR methodology. Sequencing was done on selected amplicons for determining the nim gene types. ResultsBacteroides fragilis accounted for 55.3% of the total 273 BFG members identified. Of these, 196 (71.8%) were susceptible, 43 (15.8%) intermediate and 34 (12.5%) resistant to metronidazole as determined by the E-test. nim gene was present in 101 (37%) of the total 273 isolates. Out of the 34 phenotypically resistant isolates (MIC ≥32 μg/ml), 29 harboured nim gene (Chi-square test, p < 0.0000001) but nim gene was absent in 5 (14.7%) isolates. Also, nim gene was detected in 72 (30.1%) of the 239 isolates with susceptible and intermediate metronidazole MIC. Sequencing of 20 amplicons gave a nimE gene type. ConclusionsIn view of the rising metronidazole resistance among BFG and its close association with nim genes, there is a need for implementing routine metronidazole susceptibility testing and more researches are needed to find the molecular basis of these nim genes.

Genetic diversity in the metronidazole metabolism genes nitroreductases and pyruvate ferredoxin oxidoreductases in susceptible and refractory clinical samples of Giardia lamblia

The effectiveness of metronidazole against the tetraploid intestinal parasite Giardia lamblia is dependent on its activation/inactivation within the cytoplasm. There are several activating enzymes, including pyruvate ferredoxin reductase (PFOR) and nitroreductase (NR) 1 which metabolize metronidazole into toxic forms, while NR2 on the other hand inactivates it. Metronidazole treatment failures have been increasing rapidly over the last decade, indicating genetic resistance mechanisms. Analyzing genetic variation in the PFOR and NR genes in susceptible and refractory Giardia isolates may help identify potential markers of resistance.Full length PFOR1, PFOR2, NR1 and NR2 genes from clinical culturable isolates and non-cultured clinical Giardia assemblage B samples were cloned, sequenced and single nucleotide variants (SNVs) were analyzed to assess genetic diversity and alleles.A similar ratio of amino acid changing SNVs per gene length was found for the NRs; 4.2% for NR1 and 6.4% for NR2, while the PFOR1 and PFOR2 genes had less variability with a ratio of 1.1% and 1.6%, respectively. One of the samples from a refractory case had a nonsense mutation which caused a truncated NR1 gene in one out of six alleles. Further, we found three NR2 alleles with frameshift mutations, possibly causing a truncated protein in two susceptible isolates. One of these isolates was homozygous for the affected NR2 allele. Three nsSNVs with potential for affecting protein function were found in the ferredoxin domain of the PFOR2 gene. The considerable variation and discovery of mutations possibly causing dysfunctional NR proteins in clinical Giardia assemblage B isolates, reveal a potential for genetic link to metronidazole susceptibility and resistance.

Characterisation of Trichomonas vaginalis Isolates Collected from Patients in Vienna between 2019 and 2021.

Trichomonas vaginalis (TV) is the causative agent of trichomoniasis, the most common nonviral sexually transmitted disease. TV can carry symbionts such as Trichomonas vaginalis virus (TVV) or Mycoplasma hominis. Four distinct strains of TV are known: TVV1, TVV2, TVV3, and TVV4. The aim of the current study was to characterise TV isolates from Austrian patients for the presence of symbionts, and to determine their effect on metronidazole susceptibility and cytotoxicity against HeLa cells. We collected 82 TV isolates and detected presence of TVV (TVV1, TVV2, or TVV3) in 29 of them (35%); no TVV4 was detected. M. hominis was detected in vaginal/urethral swabs by culture in 37% of the TV-positive patients; M. hominis DNA was found in 28% of the TV isolates by PCR. In 15% of the patients, M. hominis was detected in the clinical samples as well as within the respective TV isolates. In 22% of the patients, M. hominis was detected by culture only. In 11 patients, M. hominis was detected only within the respective cultured TV isolates (13%), while the swab samples were negative for M. hominis. Our results provide a first insight into the distribution of symbionts in TV isolates from Austrian patients. We did not observe significant effects of the symbionts on metronidazole susceptibility, cytotoxicity, or severity of symptoms.

Initial expression levels of nimA are decisive for protection against metronidazole in Bacteroides fragilis

ObjectivesIn the genus Bacteroides, the nim genes are resistance determinants for metronidazole, a nitroimidazole drug widely used against anaerobic pathogens. The Nim proteins are considered to act as nitroreductases. However, data from several studies suggest that the expression levels of Nim do not increase with increasing resistance which is conflicting with this notion. The impact of Nim protein levels on low-level metronidazole resistance, however, representing the early stage of induced resistance in the laboratory, has not been assessed as yet. MethodsThe nimA gene was cloned into two different plasmids and introduced into B. fragilis strain 638R. Expression levels of nimA mRNA were measured by RT-qPCR and compared to those in strain 638R harbouring plasmid pI417, the original clinical plasmid harbouring IS element IS1168 with the nimA gene. Further, metronidazole susceptibility was assessed by Etest and the activity of pyruvate:ferredoxin oxidoreductase (PFOR) was measured in all strains after induction of high-level metronidazole resistance. ResultsThe level of protection against metronidazole by nimA correleated with the level of expression of nimA mRNA. Further, the activity of PFOR in highly-resistant B. fragilis 638R was only preserved when expression levels of nimA were high. ConclusionsAlthough the development of high-level metronidazole resistance in B. fragilis strains with a nimA gene is not caused by an increase of nimA expression as compared to the less resistant parent strains, nimA expression levels might be of decisive importance in the early stage of resistance development. This has potential implications for metronidazole resistance in clinical isolates.

208 Identification of Trichomonas vaginalis 5-nitroimidazole resistance targets to inform future drug development

OBJECTIVES/GOALS: 5-nitroimidazoles are the only FDA-approved medications for T. vaginalis treatment. Resistance has been observed in 5-10% of cases, but may be rising. We aimed to delineate mechanisms of resistance in isolates of T. vaginalis using transcriptome profiling of resistant and sensitive T. vaginalis isolates. METHODS/STUDY POPULATION: T. vaginalis isolates (4 metronidazole (MTZ)-resistant were grown in triplicate in Diamond’s Trypticase-Yeast-Maltose medium. MTZ susceptibility testing confirmed MTZ MLCs of T. vaginalis isolates. Total RNA extraction was done using Trizol reagent (Invitrogen; Carlsbad; CA); according to the manufacturer’s instructions. RNA sequencing (RNAseq) and bioinformatics analyses were performed to identify significantly differentially expressed genes (DEGs) in MTZ-resistant vs. sensitive isolates. Subsequent qPCR was performed to confirm and extend RNAseq data and gene targets related to 5-nitroimidazole resistance. RESULTS/ANTICIPATED RESULTS: RNAseq identified key DEGs in MTZ-resistant vs. sensitive isolates. DEGs from MTZ-resistant isolates included those involved in metabolic pathways relevant to 5-nitroimidazole resistance such as energy production (glycolytic enzymes) and oxygen-scavenging (thioredoxin). Other DEGs included those encoding transcription factors (MYB DNA-binding protein), ribosomal proteins (30S, 40S, 50S, 60S), protein kinases (CAMK, ser/thr, CMGC), Ankyrin repeat proteins, surface proteins (Surface antigen BspA-like) and various uncharacterized hypothetical proteins. RT-qPCR experiments confirmed reduced expression of genes encoding ferredoxin (drug activation) and flavin reductase 1 (oxygen scavenging) in MTZ-resistant T. vaginalis isolates as compared to MTZ-sensitive isolates. DISCUSSION/SIGNIFICANCE: In this study, we identified several DEGs in resistant T. vaginalis isolates. Further studies with large number of isolates representing a broad range of MTZ-susceptibility patterns are needed to identify genes that may represent new targets for future drug development.

Cytidine nucleoside analog is an effective antiviral drug against Trichomonasvirus.

Trichomonas vaginalis is the causative agent of a sexually transmitted disease in humans. The virulence of the parasite depends on multiple factors, including the presence of endosymbiotic dsRNA viruses. The presence of Trichomonasviruses (TVV) was associated with more severe genital symptoms, increased proinflammatory host reactions, and modulated parasite sensitivity to metronidazole. However, no efficient antiviral drugs are available against TVV to derive isogenic TVV-positive and TVV-negative cell lines that are essential for investigations of the TVV impact on T. vaginalis biology. 7-Deaza-2'-C-methyladenosine (7d2CMA) and 2'-C-methylcytidine (2CMC) were used for TVV inhibitory assay. TVV replication was monitored using quantitative reverse transcription PCR (RT qPCR) and western blotting. Modeling of TVV1 RNA-dependent RNA polymerase (RdRp) was performed to visualize the inhibitor-RdRp interaction. Susceptibility to metronidazole was performed under aerobic and anaerobic conditions. We demonstrated that 2CMC but not 7d2CMA is a potent inhibitor of TVV replication. Molecular modeling suggested that the RdRp active site can accommodate 2CMC in the active triphosphate nucleotide form. The effect of 2CMC was shown on strains infected with a single and multiple TVV species. The optimal 2CMC concentration (10μM) demonstrated strong selectivity for TVVs over trichomonad growth. The presence of TVV has no effect on T. vaginalis metronidazole susceptibility in derived isogenic cell lines. 2CMC acts against TVVs and represents a new inhibitor against Totiviridae viruses. Our isogenic clones are now available for further studies of various aspects of T. vaginalis biology related to TVV infection.

E-Test or Agar Dilution for Metronidazole Susceptibility Testing of Helicobacter pylori: Importance of the Prevalence of Metronidazole Resistance

BackgroundA number of studies have shown that E-test overestimated the presence of Helicobacter pylori resistance compared to agar dilution.ObjectiveThe purpose of this study was to explore whether E-test could be an alternative for agar dilution to detect the metronidazole susceptibility of H. pylori.MethodE-test and agar dilution were used to assess the susceptibility of H. pylori to metronidazole, clarithromycin, and levofloxacin in 281 clinical isolates obtained from China where the resistance was high. Cohen’s kappa analysis, McNemar’s test, and essential and categorical agreement analysis were performed for these two methods.ResultsOverall, the result of the E-test showed a similar prevalence of resistance rate to all antibiotics compared with agar dilution. The essential agreement of the E-test method and agar dilution in the evaluation susceptibility of H. pylori to clarithromycin and levofloxacin was moderate at 89.0 and 79.7%, respectively, but only 45.9% for metronidazole. The results shown by a categorical agreement (CA) between the E-test and agar dilution were 100% for both clarithromycin and levofloxacin. As for metronidazole, the CA was 98.7%, no major error was identified, and the rate of a very major error was 1.8%.ConclusionE-test can be an alternative method to detect the metronidazole susceptibility of H. pylori.

Haemin deprivation renders Bacteroides fragilis hypersusceptible to metronidazole and cancels high-level metronidazole resistance.

BackgroundInfections with Bacteroides fragilis are routinely treated with metronidazole, a 5-nitroimidazole antibiotic that is active against most anaerobic microorganisms. Metronidazole has remained a reliable treatment option, but resistance does occur, including in B. fragilis.ObjectivesIn this study we tested whether haemin, a growth supplement for B. fragilis in vivo and in vitro, had an influence on the susceptibility of resistant B. fragilis strains to metronidazole. We further tested whether haemin-deprived B. fragilis would be more susceptible to oxygen and oxidative stress. Metronidazole has been described to cause oxidative stress, which we argued would be exacerbated in haemin-deprived B. fragilis because the bacteria harness haemin, and the iron released from it, in antioxidant enzymes such as catalase and superoxide dismutase.MethodsHaemin was omitted from growth media and the effect on metronidazole susceptibility was monitored in susceptible and resistant B. fragilis strains. Further, haemin-deprived B. fragilis were tested for resistance to aeration and hydrogen peroxide and the capacity for the removal of oxygen.ResultsOmission of haemin from the growth medium rendered metronidazole-resistant B. fragilis strains, including an MDR isolate from the UK, highly susceptible to metronidazole. Haemin deprivation further rendered B. fragilis highly susceptible to oxygen, which was further exacerbated in resistant strains. B. fragilis was incapable of scavenging oxygen when haemin was omitted.ConclusionsWe propose that haemin deprivation overrules resistance mechanisms by rendering B. fragilis hypersusceptible to metronidazole due to a compromised antioxidant defence. Monitoring of haemin concentrations is imperative when conducting metronidazole susceptibility testing in B. fragilis.

Bacteremia due to obligate anaerobes following large bowel surgery in a tertiary care hospital in South India

In view of the rising incidence of Anaerobic bacteremia(AB), the use of anaerobic blood culture bottles have been recommended in addition to the aerobic blood culture bottles. The need to perform antimicrobial susceptibility testing(AST) for anaerobes has become mandatory owing to increasing metronidazole resistance. The frequency of AB following large bowel surgery and the metronidazole susceptibility for members of the Bacteroides fragilis group were determined. The incidence of AB was found to be 16%. Seventeen obligate anaerobes were isolated in total, of which B. fragilis was the most common. Two of twelve isolates of B. fragilis were resistant to metronidazole.

INVESTIGATION OF CLINICAL CHARACTERISTICS OF TRICHOMONIASIS AND METRONIDAZOLE SUSCEPTIBILITY OF TRICHOMONAS STRAINS ISOLATED FROM THUA THIEN HUE

Trichomoniasis is one of the most common sexual transmitted diseases with changable clinical features. The prevalence of metronidazole resistance was different among the studies in the literature. Objectives: The aim of this study was to describe the characteristics of T. vaginalis infection and to investigate the susceptibility with metronidazole of T. vaginalis strains. Materials and methods: A cross-sectional and experimental studies were performed using the interview, the direct examination, the culture of T. vaginalis in Diamond medium, and the investigation of the metronidazole susceptibility by broth microdilution method. Results: The rate of trichomoniasis was 0.37%, in which 16.7% of patients with single status, and 76.7% of those who did not use any contraceptive methods. The common symptoms were increased vaginal discharge, itching and pain (50%); opalescent vaginal discharge (33.3%), The rate of women with asymptomatic T. vaginalis was 26.7%. The percentage of metronidazole susceptibility, intermediate and resistance to metronidazole were 73.1%, 19.2% and 7.7%, respectively. Conclusions: The study showed that T. vaginalis infection had a low rate, high proportion of asymptomatic patients and 7.7% of patients were resistant to metronidazole. Key words: T. vaginalis, metronidazole, resistant

Reduced Susceptibility to Metronidazole Is Associated With Initial Clinical Failure in Clostridioides difficile Infection.

BackgroundClinical studies have demonstrated inferior cure rates when metronidazole (MTZ) is used to treat Clostridioides difficile infection (CDI). We hypothesized that a newly identified, heme-inducible form of reduced MTZ susceptibility in C. difficile leads to higher odds of initial clinical failure in patients with CDI treated with MTZ.MethodsThis multicenter cohort study included adults diagnosed with CDI between 2017 and 2018. C. difficile isolated from stool samples underwent agar dilution MTZ susceptibility testing with incorporation of fresh heme. Blinded investigators reviewed medical records for initial clinical failure and other relevant clinical variables. Classification and regression tree (CART) analysis was used to identify the MTZ minimum inhibitory concentration (MIC) breakpoint that was predictive of initial clinical failure. Results were confirmed using univariate and multivariable logistic regression analyses to account for potential confounders.ResultsOf the 356 patients included, 72% received MTZ-based therapy and 27% experienced initial clinical failure. CART analysis identified an MTZ MIC ≥1 µg/mL above which patients had a higher rate of initial clinical failure. MTZ MICs ranged from 0.25 to 8 µg/mL (MIC50/90 = 0.25/2 µg/mL), and approximately 18% of isolates had MTZ MICs ≥1 µg/mL. In multivariable analysis, an MTZ MIC ≥1 µg/mL was an independent predictor of initial clinical failure in patients receiving an MTZ-based treatment regimen (odds ratio, 2.27 [95% confidence interval, 1.18–4.34]).ConclusionsUsing a reproducible method to determine C. difficile MICs to MTZ, a breakpoint of ≥1 µg/mL identified patients at higher risk of initial clinical failure.

Distribution of genotypes in relation to metronidazole susceptibility patterns in Trichomonas vaginalis isolated from South African pregnant women.

Reports on metronidazole resistance of Trichomonas vaginalis strains have been on the increase. This study investigated the in vitro metronidazole resistance patterns in T. vaginalis isolates obtained from South African pregnant women and the genotypes of these isolates. This study included 362 pregnant women recruited from a hospital in Durban, South Africa. The women provided self-collected vaginal swabs for the detection of T. vaginalis by culture in Diamonds media. Cultured isolates were then subjected to anaerobic susceptibility assays to metronidazole. For the genotyping assays, the actin gene was digested by HindII, MseI, and RsaI. The banding patterns obtained after digestion was used to determine the genotypes. A total of 21/362 (5.8%) pregnant women tested positive for T. vaginalis infection. Of the 21 T. vaginalis isolates tested for metronidazole susceptibility, 9.5% (2/21) had a minimum inhibitory concentration (MIC) of 4 μg/ml (resistant), 38.1% (8/21) had a MIC of 2 μg/ml (intermediate), and 52.4% (11/21) had a MIC ≤ 1 μg/ml (susceptible). The dominant genotype that was identified across the isolates was genotype G. There was no correlation between genotype harboured and metronidazole susceptibility patterns. In this study, resistance to metronidazole was observed in clinical isolates of T. vaginalis. This study did not find a correlation between genotype harboured and metronidazole susceptibility patterns. Despite the lack of association, our study provides data on an area of research that is currently lacking in our setting.

National and Regional US Antibiotic Resistance to Helicobacter pylori: Lessons From a Clinical Trial

National and Regional US Antibiotic Resistance to Helicobacter pylori: Lessons From a Clinical Trial

Heterogeneity of Helicobacter pylori Strains Isolated from Patients with Gastric Disorders in Guiyang, China.

PurposeChronic Helicobacter pylori infection causes peptic ulcers in a subpopulation of individuals and is a risk factor for the development of gastric cancer. Multiple infections and heteroresistant H. pylori contribute to poor treatment efficacy. Here, we investigated the extent of genetic diversity among H. pylori strains within a given host and its influence on the results of antibiotic (metronidazole, levofloxacin, clarithromycin, amoxicillin, and tetracycline) susceptibility testing.Materials and MethodsGastric mucosa biopsy samples were obtained from patients with gastric disorders, including 48 H. pylori positive patients, who were never previously treated for H. pylori infection. Five potential H. pylori colonies isolated from each sample were subcultured for enrichment. Enriched H. pylori colonies were identified through Gram staining and assays for urease, oxidase, and catalase. For each H. pylori monoclonal colony, the antibiotic susceptibility was assessed, genomic DNA was sequenced, and the cytotoxin-associated gene A (cagA) genotype was verified. Co-infection with multiple H. pylori strains was determined using random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR).ResultsThirteen gastric mucosa biopsy samples were positive for H. pylori. Five monoclonal strains isolated from each of these 13 patients were identified as H. pylori. RAPD-PCR indicated that intra-patient monoclonal strains of H. pylori in 10 of the 13 samples exhibited heterogeneity. Among the 13 patients, intra-patient monoclonal strains isolated from 4 patients had identical cagA genotype, whereas intra-patient monoclonal strains isolated from the other 9 patients harbored more than one cagA genotype. The antibiotic susceptibility of five intra-patient monoclonal strains from seven patients was inconsistent.ConclusionThe existence of heterogeneous H. pylori strains with resistance to different drugs and virulence were common within the gastric mucosa of an individual patient.