PCR-restriction fragment length polymorphism analysis for identification of Bacteroides spp. and characterization of nitroimidazole resistance genes

Bacteroides spp, are opportunist pathogens that cause blood and soft tissue infections and are often resistant to antimicrobial agents. We have develo ped a combined PCR-restriction fragment length polymorphism (RFLP) techniqu e to characterize the 16S rRNA gene for identification purposes and the nit roimidazole resistance (nim) gene for detection of resistance to the major antimicrobial agent used to treat Bacteroides infections: metronidazole (MT Z). 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 d iscrepancies between phenotypic and PCR-RFLP methods for eight strains were resolved by 16S rDNA sequencing in three cases, but five strains remain un identified. The presence of nim genes was indicated by PCR in 25 of 28 stra ins that exhibited reduced sensitivity to MTZ. PCR-RFLP of the nim gene pro ducts identified the four reported genes (nimA, -B, -C, and -D) and indicat ed the presence of a previously unreported nim gene in 5 strains. This nove l nim gene exhibited 75% DNA sequence similarity with nimB, These rapid, ac curate, and inexpensive methods should enable improved identification of Ba cteroides spp. and the detection of MTZ resistance determinants.