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Journal of Clinical Microbiology, September 2000, p. 3209-3213, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

PCR-Restriction Fragment Length Polymorphism Analysis for Identification of Bacteroides spp. and Characterization of Nitroimidazole Resistance Genes

Simon L. J. Stubbs,* Jon S. Brazier, Paul R. Talbot, and Brian I. Duerden

Anaerobe Reference Unit, Department of Medical Microbiology and Public Health Laboratory, University Hospital of Wales, Cardiff CF14 4XW, United Kingdom

Received 2 November 1999/Returned for modification 21 February 2000/Accepted 12 June 2000

Bacteroides spp. are opportunist pathogens that cause blood and soft tissue infections and are often resistant to antimicrobial agents. We have developed a combined PCR-restriction fragment length polymorphism (RFLP) technique to characterize the 16S rRNA gene for identification purposes and the nitroimidazole resistance (nim) gene for detection of resistance to the major antimicrobial agent used to treat Bacteroides infections: metronidazole (MTZ). PCR-RFLP analysis of 16S ribosomal (rDNA) with HpaII and TaqI produced profiles that enabled discrimination of type strains and identification of 70 test strains to the species level. The 16S rDNA PCR-RFLP identification results agreed with routine phenotypic testing for 62 of the strains. The discrepancies between phenotypic and PCR-RFLP methods for eight strains were resolved by 16S rDNA sequencing in three cases, but five strains remain unidentified. The presence of nim genes was indicated by PCR in 25 of 28 strains that exhibited reduced sensitivity to MTZ. PCR-RFLP of the nim gene products identified the four reported genes (nimA, -B, -C, and -D) and indicated the presence of a previously unreported nim gene in 5 strains. This novel nim gene exhibited 75% DNA sequence similarity with nimB. These rapid, accurate, and inexpensive methods should enable improved identification of Bacteroides spp. and the detection of MTZ resistance determinants.

* Corresponding author. Mailing address: Anaerobe Reference Unit, Public Health Laboratory, University Hospital of Wales, Heath Park, Cardiff CF14 4XW, United Kingdom. Phone: 02920-742378. Fax: 02920-744123. E-mail: Brazier{at}cf.ac.uk.

Journal of Clinical Microbiology, September 2000, p. 3209-3213, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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