Imipenem has excellent antimicrobial activity owing in part to ,-lactamase stability. We found that only 2 of over 350 Bacteroides fragilis group clinical isolates were resistant to imipenem, with an MIC of more than 16 ,ug/ml. These two isolates from the Tufts Anaerobe Laboratory (TAL) were resistant to all other P-lactam agents tested. The organisms were able to inactivate imipenem in broth cultures and contained similar P-lactamases that were able to hydrolyze carbapenems, cephamycins, cephalosporins, and penicillins. The molecular sizes of the ,-lactamases in TAL2480 and TAL3636 were estimated to be 44,000 daltons. The novel I8-lactamase contained Zn2' as a cofactor. An additional factor contributing to resistance was determined. The outer membranes of these two organisms were found to limit free diffusion of the drugs into the periplasm. This novel ,I-lactamase, associated with a barrier to drug permeation, resulted in high-grade ,-lactam drug resistance.Bacteroides fragilis is the most important anaerobic bacterial pathogen of humans. Clinical isolates generally contain ,-lactamase and as a result are frequently resistant to the cephalosporins, including the broad-spectrum cephalosporins, as well as penicillins (2,19). Only cephamycins, such as cefoxitin, and carbapenems, such as imipenem, are resistant to hydrolysis by the frequently described B. fragilis PIlactamase (2,5,14). Resistance to imipenem is rare in B. fragilis. We found that only 2 of over 350 isolates tested were resistant to imipenem. We investigated the P-lactamase activity in these two strains. MATERIALS AND METHODS Antimicrobial drugs. Standard powders were obtained from various manufacturers (19).Isolates. Bacterial identification was performed by the established method (9). Tufts Anaerobe Laboratory (TAL) isolate 2480 was isolated from the blood of a patient at the New England Deaconess Hospital, Boston, Mass. The patient died of uncontrolled sepsis while receiving cefoxitin and clindamycin. TAL3636 was isolated from a patient at the New England Medical Center Hospitals, Boston, Mass. This diabetic patient failed to respond to cefoxitin therapy and required an amputation for a foot infection.Media and incubation. The isolates were transported, stored, and incubated as previously described (10, 19). Brain heart infusion broth (Scott Laboratories, Inc., Fiskeville, R.I.) supplemented with 0.0005% hemin and 0.5% yeast extract (BHIS) was used for the liquid growth medium. BHIS was solidified, with 1.5% agar. Blood agar plates consisted of BHIS plates with 5% defibrinated sheep blood.Susceptibility testing. The MICs were determined by an agar dilution method using a Steers replicator and anaerobic chamber techniques previously described (4,19,20 for agar dilution testing. Induction of resistance was assayed by growing the organisms in broth medium with and without imipenem (5 jig/ml), cefoxitin (5 ,ug/ml), or ampicillin (20 ,ug/ml). The organisms were exposed to the drugs in both overnight and 4-h incubations.j8-Lactamase induction. ,-Lactam...

Transferable plasmids play an important role in the dissemination of clindamycin-erythromycin resistance in Bacteroides fragilis. We previously described the isolation and propetties of pBFTM10, a 14.9-kb ClnR transfer factor from B. fragilis TMP10. We also reported the isolation of a transfer-deficient deletion derivative of pBFTM10 contained in the B. fragilis-Escherichia coli shuttle vector pGAT400. In the present study we used pGAT400 and a similar shuttle vector, pGAT550, to characterize and sequence a region of pBFTM10 required for its transfer from B. fragilis to B. fragiis or E. coli recipients and for its mobilization by the broadhost-range plasmid R751 from E. coli donors to E. coli recipients. Deletion of certain BglIl restriction fragments from pBFTM1O resulted in partial or complete loss of transfer ability. TnlOOO insertions into this same region also resulted in altered transfer properties. We used the sites of TnlO00 insertions to determine the DNA sequence of the transfer region. Two potential open reading frames encoding proteins of 23.2 and 33.8 kDa, corresponding to two genes, btgA or btgB, were identified in the sequence. TnlOOO insertions within btgA or btgB or deletion of all or portions of btgA or btgB resulted in either a transfer deficiency or greatly reduced transfer from B. fragilis donors and alterations in mobilization by R751 in E. coli. A potential oriT sequence showing similarity in organization to the oriT regions of the IncP plasmids was also detected. Thus, pBFTMIO encodes and requires at least two proteins necessary for efficient transfer from B. fragilis. These same functions are expressed in E. coli and are required for mobilization by R751.There is increasing awareness that the occurrence and dissemination of drug resistance determinants in bacteria of medical importance play a significant role in the virulence of these organisms. Indeed, for Bacteroidesfragilis, one of the major obligately anaerobic bacteria involved in infections of humans, resistance to tetracycline, once rare, is now seen in more than 60% of all clinical isolates (25 addition, a region of approximately 4 kb that is essential for autonomous replication in B. fragilis has been localized (6). This leaves less than 5 kb for all remaining plasmid functions, including transfer, although it is possible that sites or functions involved in transposition and/or replication may also play a role in transfer. For this reason others have hypothesized that pBFTM10 cannot truly be self-transferable but instead must be mobilized by as yet uncharacterized products from the bacterial chromosome (21). pBFTM10 has been fused to the Escherichia coli plasmid pDG5, a pBR322 derivative containing the oriT region from the broad-host-range plasmid RP4 (6)