Inducible metronidazole resistance and nim genes in clinical Bacteroides fragilis group isolates.
Ontology highlight
ABSTRACT:
SUBMITTER: Lofmark S
PROVIDER: S-EPMC549250 | biostudies-literature | 2005 Mar
REPOSITORIES: biostudies-literature
Publications
Inducible metronidazole resistance and nim genes in clinical Bacteroides fragilis group isolates.
Löfmark Sonja S Fang Hong H Hedberg Maria M Edlund Charlotta C
Antimicrobial agents and chemotherapy 20050301 3
Nitroimidazole resistance (nim) genes were detected in 2% of 1,502 clinical Bacteroides fragilis group strains isolated from 19 European countries, and a novel nim gene was identified. High metronidazole resistance could be induced in nim-positive strains, which emphasizes the importance of acknowledging metronidazole resistance in the clinical setting. ...[more]
Similar Datasets
Project description:Two multidrug-resistant Bacteroides fragilis clinical isolates contain and express a novel nim gene, nimJ, that is not recognized by the "universal" nim primers and can confer increased resistance to metronidazole when introduced into a susceptible strain on a multicopy plasmid. HMW615, an appendiceal isolate, contains at least two copies of nimJ on its genome, while HMW616, an isolate from a patient with sepsis, contains one genomic copy of nimJ. B. fragilis NimJ is phylogenetically closer to Prevotella baroniae NimI and Clostridium botulinum NimA than to the other known Bacteroides Nim proteins. The predicted protein structure of NimJ, based on fold recognition analysis, is consistent with the crystal structures derived for known Nim proteins, and specific amino acid residues important for substrate binding in the active site are conserved. This study demonstrates that the "universal" nim primers will not detect all nim genes with the ability to confer metronidazole resistance, but nimJ alone cannot account for the very high metronidazole MICs of these resistant clinical isolates.
| S-EPMC3719759 | biostudies-literature
Project description:Background:Bacteroides fragilis is the most common anaerobic pathogen isolated from surgical site infections (SSIs). Metronidazole resistance is increasing and the mechanisms of resistance are not clear in some isolates. The aim of the present study was to investigate the metronidazole susceptibility prevalence, and detect nim genes in B. fragilis isolates from SSIs. Methods: This study included 100 surgery patients with signs and symptoms indicative of SSIs. Syringe aspiration of the infected site was used to collect specimens. All specimens were cultured on BBA (Brucella blood agar), KVLB (kanamycin-vancomycin laked blood), and BBE (Bacteroides bile esculin) agar. The MIC (minimum inhibitory concentration) of metronidazole was determined by the agar dilution method according to the Clinical and Laboratory Standard Institute (CLSI). Then the PCR method was used to determine the presence of the nim gene. Results: In the present study, 26 B. fragilis were isolated from 100 SSIs specimens. Eight isolates were metronidazole resistant; the metronidazole MIC was 32 µg/mL for 7 isolates and 64 µg/mL for one isolate. All isolates were nim gene negative. Conclusion: The emergence of metronidazole-resistant B. fragilis limits the application of this drug for treatment and prophylaxis of SSIs. Thus, rapid identification of metronidazole-resistant B. fragilis is essential to restrict inappropriate, superfluous administration. In spite of various metronidazole resistance mechanisms other than that depending on the nim gene, detection of nim by PCR is unsuitable for identifying resistant isolates. Therefore, phenotypic methods are better to screen for and identify metronidazole-resistant B. fragilis.
| S-EPMC5564006 | biostudies-other
Project description:Members of the genus Bacteroides, mainly Bacteroides fragilis, can cause severe disease in man, especially after intestinal perforation in the course of abdominal surgery. Treatment is based on a small number of antibiotics, including metronidazole, which has proved to be highly reliable throughout the last 40 to 50 years. Nevertheless, metronidazole resistance does occur in Bacteroides and has been mainly attributed to Nim proteins, a class of proteins with a suggested nitroreductase function. Despite the potentially high importance of Nim proteins for human health, information on the expression of nim genes in B. fragilis is still lacking. It was the aim of this study to demonstrate expression of nim genes in B. fragilis at the protein level and, furthermore, to correlate Nim levels with the magnitude of metronidazole resistance. By the application of 2D gel electrophoresis, Nim proteins could be readily identified in nim-positive strains, but their levels were not elevated to a relevant extent after induction of resistance with high doses of metronidazole. Thus, the data herein do not provide evidence for Nim proteins acting as nitroreductases using metronidazole as a substrate, because no correlation between Nim levels and levels of metronidazole resistance could be observed. Furthermore, no evidence was found that Nim proteins protect B. fragilis from metronidazole by sequestering the activated antibiotic.
| S-EPMC5215050 | biostudies-literature
Project description:Bacteroides fragilis is a human gut commensal and an opportunistic pathogen causing anaerobic abscesses and bacteraemias which are treated with metronidazole (Mtz), a DNA damaging agent. This study examined the role of the DNA repair protein, RecA, in maintaining endogenous DNA stability and its contribution to resistance to Mtz and other DNA damaging agents. RT-PCR of B. fragilis genomic DNA showed that the recA gene was co-transcribed as an operon together with two upstream genes, putatively involved in repairing oxygen damage. A B. fragilis recA mutant was generated using targeted gene inactivation. Fluorescence microscopy using DAPI staining revealed increased numbers of mutant cells with reduced intact double-stranded DNA. Alkaline gel electrophoresis of the recA mutant DNA showed increased amounts of strand breaks under normal growth conditions, and the recA mutant also showed less spontaneous mutagenesis relative to the wild type strain. The recA mutant was sensitive to Mtz, ultraviolet light and hydrogen peroxide. A B. fragilis strain overexpressing the RecA protein exhibited increased resistance to Mtz compared to the wild type. This is the first study to show that overexpression of a DNA repair protein in B. fragilis increases Mtz resistance. This represents a novel drug resistance mechanism in this bacterium.
| S-EPMC3025348 | biostudies-literature
Project description:Bacteroides fragilis group (BFG) are the most frequently recovered anaerobic bacteria from human infections, and resistance to frontline antibiotics is emerging. In the absence of routine antimicrobial susceptibility testing (AST) for BFG in most clinical settings, we assessed the utility of clinical and modern genomics tools to determine BFG species-level identification and resistance patterns. A total of 174 BFG clinical isolates supplemented with 20 archived carbapenem-resistant B. fragilis sensu stricto (BFSS) isolates underwent antimicrobial susceptibility testing, MALDI-ToF mass-spectrometry, and whole-genome sequencing (WGS). Bruker BioTyper and VITEK-MS MALDI-ToF systems demonstrated accurate species-level identifications (91% and 90% agreement, respectively) compared to average nucleotide identity (ANI) analysis of WGS data. Distinct β-lactamase gene profiles were observed between BFSS and non-fragilis Bacteroides species, with significantly higher MICs to piperacillin-tazobactam in B. vulgatus and B. thetaiotaomicron relative to BFSS (P ≤ 0.034). We also uncovered phylogenetic diversity at the genomospecies level between division I and division II BFSS (ANI <0.95) and demonstrate that division II BFSS strains harbor an increased capacity to achieve carbapenem resistance through chromosomal activation of the CfiA carbapenemase. Finally, we report that CfiA detection by the Bruker BioTyper Subtyping Module accurately detected carbapenem resistance in BFSS with positive and negative percent agreement of 94%/90% and 95%/95% compared to ertapenem and meropenem susceptibility, respectively. These comparative analyses indicate that resistance mechanisms are distinct at both the phenotypic and genomic level across species within the BFG and that modern MALDI-ToF identification systems can be used for accurate species-level identification and resistance prediction of the BFG. IMPORTANCE Anaerobic infections present unique challenges in terms of detecting and identifying the etiologic agent and selecting the optimal antimicrobial therapy. Antimicrobial resistance is increasing in anaerobic pathogens, and it is critical to understand the prevalence and mechanisms of resistance to commonly prescribed antimicrobial therapies. This study uses comparative genomics to validate clinical tools for species-level identification and phenotypic resistance prediction in 194 isolates of Bacteroides fragilis group (BFG) bacteria, which represent the most commonly isolated organisms among anaerobic infections. We demonstrate species-specific patterns in antimicrobial resistance and validate new strategies for species-level organism identification and phenotypic resistance prediction in a routine clinical laboratory setting. These findings expand our understanding and management of anaerobic infections and justify further investigations into the molecular basis for species-specific resistance patterns observed within this study.
| S-EPMC8764542 | biostudies-literature
Project description:The anaerobic gut bacteria and opportunistic pathogen Bacteroides fragilis can cause life-threatening infections when leaving its niche and reaching body sites outside of the gut. The antimicrobial metronidazole is a mainstay in the treatment of anaerobic infections and also highly effective against Bacteroides spp. Although resistance rates have remained low in general, metronidazole resistance does occur in B. fragilis and can favor fatal disease outcomes. Most metronidazole-resistant Bacteroides isolates harbor nim genes, commonly believed to encode for nitroreductases which deactivate metronidazole. Recent research, however, suggests that the mode of resistance mediated by Nim proteins might be more complex than anticipated because they affect the cellular metabolism, e.g., by increasing the activity of pyruvate:ferredoxin oxidoreductase (PFOR). Moreover, although nim genes confer only low-level metronidazole resistance to Bacteroides, high-level resistance can be much easier induced in the laboratory in the presence of a nim gene than without. Due to these observations, we hypothesized that nim genes might induce changes in the B. fragilis proteome and performed comparative mass-spectrometric analyses with B. fragilis 638R, either with or without the nimA gene. Further, we compared protein expression profiles in both strains after induction of high-level metronidazole resistance. Interestingly, only few proteins were repeatedly found to be differentially expressed in strain 638R with the nimA gene, one of them being the flavodiiron protein FprA, an enzyme involved in oxygen scavenging. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter, the observed changes were not only less numerous but also less specific. Both resistant strains, however, displayed a reduced capability of scavenging oxygen. Susceptibility to metronidazole could be widely restored in resistant 638R without nimA by supplementing growth media with ferrous iron sulfate, but not so in resistant 638R with the nimA gene. Finally, based on the results of this study, we present a novel hypothetic model of metronidazole resistance and NimA function.
| S-EPMC10102347 | biostudies-literature
Project description:Bacteroides fragilis is a commensal of the human gut but can also cause severe infections when reaching other body sites, especially after surgery or intestinal trauma. Bacteroides fragilis is an anaerobe innately susceptible to metronidazole, a 5-nitroimidazole drug that is prescribed against the majority of infections caused by anaerobic bacteria. In most of the cases, metronidazole treatment is effective but a fraction of B. fragilis is resistant to even very high doses of metronidazole. Metronidazole resistance is still poorly understood, but the so-called nim genes have been described as resistance determinants. They have been suggested to encode nitroreductases which reduce the nitro group of metronidazole to a non-toxic aminoimidazole. More recent research, however, showed that expression levels of nim genes are widely independent of the degree of resistance observed. In the search for an alternative model for nim-mediated metronidazole resistance, we screened a strain carrying an episomal nimA gene and its parental strain 638R without a nim gene for physiological differences. Indeed, the 638R daughter strain with the nimA gene had a far higher pyruvate-ferredoxin oxidoreductase (PFOR) activity than the parental strain. High PFOR activity was also observed in metronidazole-resistant clinical isolates, either with or without a nim gene. Moreover, the strain carrying a nimA gene fully retained PFOR activity and other enzyme activities such as thioredoxin reductase (TrxR) after resistance had been induced. In the parental strain 638R, these were lost or very strongly downregulated during the development of resistance. Further, after induction of high-level metronidazole resistance, parental strain 638R was highly susceptible to oxygen whereas the daughter strain with a nimA gene was hardly affected. Ensuing RT-qPCR measurements showed that a pathway for iron import via hemin uptake is downregulated in 638R with induced resistance but not in the resistant nimA daughter strain. We propose that nimA primes B. fragilis toward an alternative pathway of metronidazole resistance by enabling the preservation of normal iron levels in the cell.
| S-EPMC9218692 | biostudies-literature
Project description:Bacteroides fragilis constitutes a significant part of the normal human gut microbiota and can also act as an opportunistic pathogen. Antimicrobial resistance (AMR) and the prevalence of AMR genes are increasing, and prediction of antimicrobial susceptibility based on sequence information could support targeted antimicrobial therapy in a clinical setting. Complete identification of insertion sequence (IS) elements carrying promoter sequences upstream of resistance genes is necessary for prediction of AMR. However, de novo assemblies from short reads alone are often fractured due to repeat regions and the presence of multiple copies of identical IS elements. Identification of plasmids in clinical isolates can aid in the surveillance of the dissemination of AMR, and comprehensive sequence databases support microbiome and metagenomic studies. We tested several short-read, hybrid and long-lead assembly pipelines by assembling the type strain B. fragilis CCUG4856T (=ATCC25285=NCTC9343) with Illumina short reads and long reads generated by Oxford Nanopore Technologies (ONT) MinION sequencing. Hybrid assembly with Unicycler, using quality filtered Illumina reads and Filtlong filtered and Canu-corrected ONT reads, produced the assembly of highest quality. This approach was then applied to six clinical multidrug-resistant B. fragilis isolates and, with minimal manual finishing of chromosomal assemblies of three isolates, complete, circular assemblies of all isolates were produced. Eleven circular, putative plasmids were identified in the six assemblies, of which only three corresponded to a known cultured Bacteroides plasmid. Complete IS elements could be identified upstream of AMR genes; however, there was not complete correlation between the absence of IS elements and antimicrobial susceptibility. As our knowledge on factors that increase expression of resistance genes in the absence of IS elements is limited, further research is needed prior to implementing AMR prediction for B. fragilis from whole-genome sequencing.
| S-EPMC6927303 | biostudies-literature
Project description:Metronidazole is the most commonly used antimicrobial for Bacteroides fragilis infections and is recommended for prophylaxis of colorectal surgery. Metronidazole resistance is increasing and the mechanisms of resistance are not clear.A transposon mutant library was generated in B. fragilis 638R (BF638R) to identify the genetic loci associated with resistance to metronidazole.Thirty-two independently isolated metronidazole-resistant mutants had a transposon insertion in BF638R_1421 that encodes the ferrous transport fusion protein (feoAB). Deletion of feoAB resulted in a 10-fold increased MIC of metronidazole for the strain. The metronidazole MIC for the feoAB mutant was similar to that for the parent strain when grown on media supplemented with excess iron, suggesting that the increase seen in the MIC of metronidazole was due to reduced cellular iron transport in the feoAB mutant. The furA gene repressed feoAB transcription in an iron-dependent manner and disruption of furA resulted in constitutive transcription of feoAB, regardless of whether or not iron was present. However, disruption of feoAB also diminished the capacity of BF638R to grow in a mouse intraperitoneal abscess model, suggesting that inorganic ferrous iron assimilation is essential for B. fragilis survival in vivo.Selection for feoAB mutations as a result of metronidazole treatment will disable the pathogenic potential of B. fragilis and could contribute to the clinical efficacy of metronidazole. While mutations in feoAB are probably not a direct cause of clinical resistance, this study provides a key insight into intracellular metronidazole activity and the link with intracellular iron homeostasis.
| S-EPMC4164142 | biostudies-literature
Project description:Bacteroides fragilis is an anaerobic commensal in the human gut which can act as opportunistic pathogen when leaving its intestinal niche to reach other body sites. Bacteroides infections have a high lethality and must be treated by antimicrobial chemotherapy. Metronidazole is one of the most frequently administered antimicrobials in the treatment of Bacteroides infections and is highly reliable. However, metronidazole resistance does occur, favoring fatal disease outcomes. Most of the resistant isolates harbor a nim gene (12 are currently known, i.e. nimA to nimL), a transferable resistance determinant for metronidazole. Previous research suggested that Nim proteins might affect the cellular physiology by changing the activity of key enzymes like pyruvate:ferredoxin oxidoreductase (PFOR). In this study we wanted to assess the impact of the nimA gene on protein expression in a standard B. fragilis isolate, 638R, and compared overall protein expression in 638R with and without a nim gene and with the nimA gene in a proteomic study. Further, high-level metronidazole resistance was induced in both strains and the protein expression profiles of resulting resistant daughter strains were also compared with their respective parent strains. We found that comparably few proteins displayed altered expression in 638R with the nimA gene, but flavodiiron protein FprA was repeatedly found upregulated. FprA is often found in anaerobes and reduces molecular oxygen to water and/or nitric oxide to nitrous oxide. After induction of metronidazole resistance, a far higher number of proteins were found to be differentially expressed in 638R without nimA than in 638R with nimA. In the former, factors for the import of hemin were strongly downregulated, indicating impaired iron import, whereas in the latter the observed changes were not only less numerous but also less specific. Based on the results of this study we present a novel hypothetic model of metronidazole resistance and Nim function.
2023-03-20 | PXD039809 | Pride