Significance: Bacteroides fragilis (BF), an obligatory anaerobic bacterium that is normally a gut commensal, can become an opportunistic pathogen and cause serious infections. It is the major Bacteroides species isolated from human anaerobic infections (80%). Metronidazole (MET) is among the most commonly used antibiotics against BF. Unfortunately, MET resistance among BF is becoming more prevalent and MET resistant (MET-R) strains have resulted in amputations, sepsis and death. While MET resistance in BF has been canonically ascribed to the presence of the nim gene (coding for the NIM protein which prevents the formation of the toxic MET molecule), our results as well as other reports indicate otherwise and suggest that MET resistance is multifactorial. Innovation: This project seeks to identify mechanisms that confer MET resistance in BF using an integrated approach combining classical genetics, Next Generation Sequencing technology and molecular manipulation to relate function to specific genes. Our lab has become skilled in using these techniques for BF research and has a strong global network of colleagues who will send us clinical MET-R BF isolates. Research strategy:
In Aim 1 A we will study transcriptome changes among spontaneous, sequential MET-R mutants.
In Aim 1 B, we will identify gene(s) responsible for MET resistance in a saturated transposon mutant library. The genes identified as likely candidates for involvement in MET resistance will be targeted for molecular manipulation and either deleted or overexpressed, as appropriate (Aim 1C).
In Aim 2 A, we will study the clinical MET-R isolates with genomic sequencing and RNA-SEQ based transcriptome analysis with subsequent comparisons with genomes and transcriptomes from other clinical and lab isolates. We will also use an unbiased approach to find MET resistance-associated genes by introducing genomic libraries made from clinical MET-R strains into the MET susceptible lab strain, BF638R.
In Aim 2 C, we will demonstrate functional associations of these genes by molecular manipulation (as in Aim 1C).
In Aim 3 we will focus on nim-mediated MET resistance and determine whether certain nim alleles are more likely to confer the high MICs seen in clinically resistant strains (Aim 3A) and whether nim might be transferred between strains via a conjugative transposon mobile element (Aim 3B). Impact: The proposed work will provide important NGS data for the Bacteroides research community and suggest a new integrated approach with which to study this pathogen. The data regarding nim gene activity as well as involvement of other key genes will be used to develop new rapid diagnostic tests for MET resistance. In addition, identifying the multiple factors in MET resistance will provide guidance t drug developers in designing therapy.

Public Health Relevance

Bacteroides fragilis (BF), normally a benign microbe in the human gut, can become a severe, virulent and dangerous pathogen. Metronidazole (MET) is the preferred antibiotic worldwide to treat BF;however, increasing resistance is seen and the mechanisms are not well understood and therefore resistance is not easily predicted. This project proposes an integrated approach using Next-Generation Sequencing analysis and sophisticated molecular techniques to study MET resistance in BF and the data generated will lead to better diagnostic techniques and guidance for drug developers.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
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Baqar, Shahida
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Brentwood Biomedical Research Institute
Los Angeles
United States
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Veeranagouda, Yaligara; Husain, Fasahath; Boente, Renata et al. (2014) Deficiency of the ferrous iron transporter FeoAB is linked with metronidazole resistance in Bacteroides fragilis. J Antimicrob Chemother 69:2634-43
Husain, Fasahath; Veeranagouda, Yaligara; Boente, Renata et al. (2014) The Ellis Island Effect: A novel mobile element in a multi-drug resistant Bacteroides fragilis clinical isolate includes a mosaic of resistance genes from Gram-positive bacteria. Mob Genet Elements 4:e29801
Veeranagouda, Yaligara; Husain, Fasahath; Tenorio, Elizabeth L et al. (2014) Identification of genes required for the survival of B. fragilis using massive parallel sequencing of a saturated transposon mutant library. BMC Genomics 15:429
Veeranagouda, Yaligara; Husain, Fasahath; Wexler, Hannah M (2013) Transposon mutagenesis of Bacteroides fragilis using a mariner transposon vector. Anaerobe 22:126-9
Husain, Fasahath; Veeranagouda, Yaligara; Hsi, Justin et al. (2013) Two multidrug-resistant clinical isolates of Bacteroides fragilis carry a novel metronidazole resistance nim gene (nimJ). Antimicrob Agents Chemother 57:3767-74
Brook, Itzhak; Wexler, Hannah M; Goldstein, Ellie J C (2013) Antianaerobic antimicrobials: spectrum and susceptibility testing. Clin Microbiol Rev 26:526-46
Veeranagouda, Yaligara; Husain, Fasahath; Wexler, Hannah M (2012) Transposon mutagenesis of the anaerobic commensal, Bacteroides fragilis, using the EZ::TN5 transposome. FEMS Microbiol Lett 333:94-100