Beta-lactamase-mediated imipenem resistance in Bacteroides fragilis

Abstract

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...

Resistance to -Lactam Antibiotics in Bacteroides Spp.

“…Also, P-lactamase-mediated imipenem resistance in B. fragilis has been shown to be associated with a barrier to drug permeation, as determined by crypticity measurements [42]. In contrast, a recent study showed no correlation between the levels of imipenem resistance of B. fragilis isolates and crypticity or other permeability measurements that take into account kinetic factors involved in drug permeation (personal unpublished results).…”

“…This enzyme has been designated a member of Bush group 2d [63]. Also, atypical p-lactamases from strains of Bacteroides spp., such as B. fragilis strain TAL4170 and B. uniformis strain 2986, have been reported that inactivate cefoxitin, show diverse PI values and exhibit various levels of susceptibility to clavulanic acid [20, 211. A distinct class of B. fragilis p-lactamase that inactivated a wide range of p-lactam substrates usually considered stable to hydrolysis, including cephamycins and carbapenems, was reported in 1986 by Cuchural et al [42]. These enzymes were inhibited by the ion chelator EDTA, and zinc ions completely reversed this inhibition.…”

“…[88] reported a potent P-lactamase produced by a B. fragilis strain with a similar substrate profile to that described by Cuchural et al [42], i.e., capable of hydrolysing cephamycin derivatives and imipenem, and not susceptible to clavulanic acid. However, EDTA inhibition was not mentioned in this report and this enzyme has been assigned to group 4 of the Bush classification scheme [63].…”

Genotypic identification of two groups within the species Bacteroides fragilis by ribotyping and by analysis of PCR-generated fragment patterns and insertion sequence content

“…Thirty-eight nonrandomly collected cfiA-positive carbapenem-resistant (Cpm r ) or carbapenem-susceptible (Cpm s ) clinical isolates were included in this study (Table 1): 34 strains, designated BFr, some of which have been described previously (21,22), were isolated in France since 1983 or in Spain since 1992 (the latter, comprising 5 strains, were kindly provided by C. Betriu); 2 strains, TAL3636 (not shown in Table 1) and TAL2480, were isolated in the United States (7,37); one strain, B119, was isolated in the United Kingdom (9); and one strain, KSB1468, was isolated in Sweden (13). The strains were grown in Wilkins-Chalgren (WC; Oxoid) liquid medium or on WC agar plates in an anaerobic atmosphere produced by a gas-generating kit (Anaerobic System BR38; Oxoid).…”

“…Production of CepA leads to resistance to most ␤-lactam antibiotics, which are otherwise efficient in the treatment of B. fragilis infections, with the exception of the cephamycins, the carbapenems, and the ␤-lactamase inhibitors. These last three groups of compounds, and most typically the carbapenems imipenem and meropenem, remain without effect against strains producing the metallo-␤-lactamase CfiA (7,9,21,44). The corresponding gene can be silent or expressed, depending upon the promoter which drives its transcription (21,22).…”